CN110564821B

CN110564821B - RNA in-situ hybridization optimization method for cucumber seedling gene localization research

Info

Publication number: CN110564821B
Application number: CN201910751470.4A
Authority: CN
Inventors: 张文娜; 胡茜; 高丽红
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2019-08-15
Filing date: 2019-08-15
Publication date: 2020-10-02
Anticipated expiration: 2039-08-15
Also published as: WO2021027866A1; CN110564821A

Abstract

The invention relates to an RNA in-situ hybridization optimization method for cucumber seedling gene localization research. Firstly, according to the characteristics of cucumber seedling plant materials, improving the methods of plant culture, sampling and fixation, thereby obtaining a complete plant structure containing target tissues; secondly, operation methods such as section preparation, hybridization pretreatment, hybridization incubation and sealing, chromogenic reaction microscopic examination and the like are optimized in a targeted manner, so that the reliability, accuracy and specificity of a gene positioning result are improved; finally, a treatment scheme suitable for the mature tissue of the cucumber seedling after the color reaction is terminated is designed in a targeted manner so as to eliminate the interference of cell contents and directly observe the distribution of hybridization signals. The invention is not only used for improving the sensitivity and the accuracy of the experimental result, but also reduces the economic cost by improving the utilization rate of the medicine, reducing the consumption of experimental articles, increasing the yield of reaction products, saving the labor cost and the like, thereby being a set of scientific research method which gives consideration to scientificity, innovativeness and practicability.

Description

RNA in-situ hybridization optimization method for cucumber seedling gene localization research

Technical Field

The invention relates to the technical field of plant biology, in particular to an RNA in-situ hybridization optimization method for cucumber seedling gene localization research.

Background

Cucumber (Cucumis sativus L.) is a main vegetable crop in China and also an important economic crop. According to the statistical data of the world Food and Agriculture Organization (FAO), the annual yield of the Chinese cucumber accounts for 48.3% of the annual yield of the cucumber in the world in 1961, and the proportion reaches 77.5% in 2017, wherein China is the most important cucumber cultivation place in the world. Therefore, the research on the physiological cultivation mechanism and the gene function characteristics of the cucumber has important significance on the construction of modern agriculture, the progress of cultivation management modes and the breeding of high-quality new varieties. With the progress of genomics and bioinformatics, the whole genome information of cucumber is finally decoded in 2009 (Huang et al 2009), however, most of the decoded nucleotide sequences are new genes with missing annotation information. On one hand, the disclosed whole genome information provides great convenience for the research of the molecular mechanism of cucumber; on the other hand, due to the low genetic transformation rate of cucumber, the rapid screening of key genes from a large number of candidate genes also becomes a new challenge.

The function and location of the gene are closely related. Cucumber belongs to an organism with multiple cell types, and comprises various tissues and organs formed by cells with different differentiation types, different tissue parts bear different physiological functions, and genes expressed in the same tissue possibly bear similar or connected functions. For example, epidermal cells of plant root hair, which are primarily responsible for absorbing water and mineral nutrients from the soil; the plant vascular bundle tissue is responsible for transporting nutrient substances and metabolic substances required by the growth and development of plants. Therefore, the research on gene functions of gene positioning and the screening of key genes have guiding significance.

The current mainstream plant tissue level gene localization research methods comprise an in situ hybridization method, a GUS histochemical localization method and a GFP fluorescent labeling localization method. However, the genetic transformation efficiency of cucumber is still low, and only the in situ hybridization method in the three methods does not need to construct a cucumber transgenic interference line, so that the method is more suitable for the gene localization research of the tissue level of cucumber at the present stage. In situ hybridization techniques originated in the middle of the last century, and RNA in situ hybridization methods applied to gene mapping of plant tissues were mainly based on the reports of DaveJackson and Cox Kathleen H (JACKSON and DAVID 1991; Meyerowitz and M1987). The basic principle of the nested system is that DIG (digoxin) -antisense probe complementary to mRNA is hybridized, then AP (alkaline phosphatase) capable of being coupled with DIG is added as a secondary antibody, and the AP and two chromogenic substrates NBT (tetrazolium blue) and BCIP (5-bromo-4-chloro-3-indole-phosphate) are subjected to chromogenic reaction to generate purple red. Different plant materials have different characteristics, and particularly, the plant materials of the mature tissues and the meristematic tissues of the plants have larger difference in the application aspect of the in situ hybridization technology. The test methods of Dave Jackson and Cox Kathleen H are more suitable for the study of plant meristems. In recent years, there are some In Situ Hybridization systems (In Situ Hybridization protocols) -Benfey Lab 2019; Cheng et al 2018) optimized for plants, but none of these In Situ Hybridization systems can be directly applied to the gene localization research of mature tissues of cucumber seedlings.

The existing in situ hybridization technology system applied to plants is suitable for meristematic tissues of plants (such as leaf bud development, pistil differentiation, embryo or ovule development and the like), and is also suitable for mature tissues of most plants (such as roots of apple seedlings), wherein plant materials are thick and strong, and become hard after meeting a stationary liquid or have high lignification degree. The roots, stems and other parts of cucumber seedlings are too young and tender, hybrid molecular signals of mature tissues are different from those of meristems, and the following problems occur when the conventional system is directly applied:

(1) plant material cultivation: the root system of cucumber seedling cultivated by the traditional substrate of turf vermiculite and perlite can be adhered with a plurality of substrate impurities which are difficult to remove, but the impurities cannot be completely removed by violent flushing, and the tissue structure of the root system of cucumber can be damaged;

(2) preparing a gene probe: in the traditional system, a target gene cDNA fragment is connected with pGEMT or pGEMTeasy, and then an in vitro transcription template is obtained by recovering an enzyme digestion linear vector product, however, the method not only increases the economic cost, but also the yield of the template is directly influenced by the connection efficiency and the enzyme digestion efficiency;

(3) sampling and fixing plant materials: the stem and root system of the cucumber seedling are very young and tender, the density is low, and the cucumber seedling is difficult to precipitate after being vacuumized. The traditional sample cutting mode, the fixing solution formula, the gradient ethanol dehydration system and the paraffin embedding mode are not suitable, so that tissues are shrunk and damaged, and the structure is incomplete (as shown in figure 1);

(4) preparation of hybridization sections: the traditional system is not optimized aiming at the step, and the step has important influence on the in-situ hybridization result of the mature tissue of the later-stage cucumber seedling;

(5) hybridization pretreatment: the traditional system does not optimize the sample preservation processed in the step, and a plurality of reagent solutions are used for a plurality of times and are greatly consumed at the stage;

(6) hybridization incubation mounting: the traditional system is not optimized aiming at the step, and the technical method of the step has direct influence on the accuracy of the gene positioning result of the mature tissue of the cucumber seedling;

(7) microscopic examination of color reaction: the traditional system is not optimized aiming at the step, and the step has direct influence on the specificity of the gene in the positioning of the mature tissue of the cucumber seedling;

(8) and (3) reaction termination post-treatment: the traditional system requires TE buffer solution to stop color reaction, then xylene and ethanol are used for gradient rinsing, or clear water is used for stopping reaction and then directly photographing is carried out, and hybrid molecule signals of mature tissues of cucumber seedlings are distributed in a 'dot' mode and are different from hybrid molecule signals distributed in a 'surface' mode in meristematic tissues, so that the hybrid signals can be interfered by a large number of cell contents and cannot be clearly seen, and can be easily and directly decolored by ethanol or xylene rinsing (as shown in figure 1).

The method requires a new in-situ hybridization optimization scheme suitable for mature tissues of cucumber seedlings, and solves the technical defects in the steps of cucumber seedling material source, sampling, fixing, probe preparation, hybridization process, hybridization color development termination post-treatment and the like in the application of the existing system.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide an RNA in-situ hybridization optimization method for cucumber seedling gene localization research.

In order to achieve the above purposes, the technical scheme adopted by the invention is as follows:

an RNA in-situ hybridization optimization method for cucumber seedling gene localization research comprises the following steps:

step 1, culturing a test material, wherein the test material is cucumber seedlings;

the overground tissue material of the cucumber seedling has no special requirement on the culture condition, and the underground tissue material is cultured by tissue culture, water culture, sand culture and soil culture; the soil culture is fine soil culture;

step 2, sampling and fixing a test material;

sampling on ice, cutting stems of cucumber seedlings with the length of 3-4 cm and root segments containing lateral roots as plant samples, embedding to obtain embedded samples, and performing block repairing and cutting on the embedded samples (the section size of a paraffin block is based on the section of a tissue sample, and the edge) to obtain the tissue sample;

step 3, preparing a probe of the target gene, and purifying the probe;

the transcription template of the probe consists of a template fragment and a T7/Sp6 promoter sequence, the connection directions of a sense probe and an antisense probe are opposite to that of the promoter, the sense probe is a negative control of a target gene, the antisense probe is a complementary sequence of the target gene, the antisense probe is combined with a target gene to display the position of the target gene, and the sense probe and the antisense probe are transcription template in vitro transcription products with the target gene;

selecting a target gene fragment with GG initiation and GC content of 40-70% as a template fragment according to the mRNA sequence length of a target gene and a fluorescent quantitative qPCR result;

step 4, preparing a hybridization slice;

preparing a paraffin section from the tissue sample obtained in the step 2 by a slicer, screening the paraffin section, and spreading and drying the paraffin section in sequence to obtain a hybrid section;

the screened paraffin sections are gently placed in RNase-free water at 37 ℃ for spreading, the paraffin sections are completely spread after being softened, the color is changed from white to almost transparent, the paraffin sections are gently dragged and spread on a glass slide by a brush pen, bubbles are removed by slight pressure of the brush pen, and water is absorbed by slight pressure of absorbent paper, so that the time for drying the paraffin sections is shortened, the effect of drying the paraffin sections is improved, later-stage separation of samples from the glass slide is avoided, and the hybridized sections are obtained;

in the slicing process, wax stains on the blade are checked and cleaned in time, and sample fragmentation caused by scratches is avoided;

step 5, hybridization pretreatment;

dewaxing and decoloring the hybrid slices, and treating the protein wrapping the target molecules to fully expose the target molecules; storing the finally obtained hybrid slice sample in a refrigerator at 4 ℃, and placing the hybrid slice sample on a dyeing frame in a dyeing cylinder sealed by a sealing film with a small amount of absolute ethyl alcohol added at the bottom;

step 6, hybridization incubation mounting:

performing first hybridization incubation on the hybridization slice sample obtained in the step 5 by using a primary antibody, and then performing second hybridization incubation by using a secondary antibody, wherein the primary antibody is a DIG-11-UTP probe, and the secondary antibody is DIG-AP; rinsing the incubated hybridization slice sample by using an RNA hybridization solution containing an antibody, and then sealing by using the RNA hybridization solution containing the antibody for incubation;

the RNA hybridization solution sealing, namely sample sealing, requires that a sample after hybridization pretreatment must be completely soaked in the RNA hybridization solution, once bubbles are generated, the sample at the position is not contacted with an antibody, and therefore the accuracy of a gene positioning result is influenced;

step 7, microscopic examination of color reaction:

the gene with high gene expression abundance at the cucumber seedling stage, namely the gene with the CQ value of 15-25 of the fluorescent quantitative PCR and the CQ value of the internal reference gene of 15, should be subjected to microscopic examination once within 6h from the beginning of the color reaction to check a hybridization signal; genes with general expression abundance, namely genes with CQ value of more than or equal to 25 and less than or equal to 30 of the fluorescence quantitative PCR and CQ value of the internal reference gene of 15, are subjected to microscopic examination 2 times each day in the morning, afternoon or evening on the next day;

screening out light as much as possible during microscopic examination, placing the hybridization slice sample subjected to the sealing in the step 6 in NBT/BCIP chromogenic substrate buffer solution for separation, making a temporary mounting by using the NBT/BCIP chromogenic substrate buffer solution, then placing under a microscope for examination of a hybridization signal, and if clear hybridization signal (mauve) molecules are observed, terminating the chromogenic reaction; otherwise, the sample is washed again by NBT/BCIP chromogenic substrate buffer solution, and then the two glass slides are bonded again and put back into the Cooprin cup for continuous reaction;

and 8, reaction termination post-treatment:

for tissue samples where hybridization signals were observed, the color reaction was stopped with clear water, then left to air dry at room temperature, and finally each slide was directly coated with 90 μ l of neutral resin: and sealing the slices with mixed solution of xylene in a ratio of 1:1, and permanently storing experimental results.

On the basis of the technical scheme, in the step 2, the embedding process is as follows:

quickly placing the taken plant sample into precooled FAA stationary liquid, vacuumizing for 18-20 min by using a vacuum pump, slowly introducing air until the atmospheric pressure is restored, replacing new FAA stationary liquid and shaking in a horizontal shaking table at 4 ℃ overnight, wherein the plant sample treated in the way can be stored for 1 week at 4 ℃ in a short period;

dehydrating the plant sample: dehydrating with RNase-free water in ethanol at 4 deg.C in gradient of 50%, 70%, 85%, 95%, 100%, from low to high concentration, each gradient being supplemented with 1-2 earpick of eosin stain for distinguishing paraffin from the sample during slicing until the plant sample is stained pink, dehydrating for 35min in each gradient, and finally overnight with 100% ethanol;

performing xylene replacement on ethanol in a horizontal shaking table at normal temperature, sequentially comprising 100% ethanol, 50% ethanol + 50% xylene, 100% xylene and 100% xylene, wherein the replacement time of each gradient is 30min, and the last 100% xylene is overnight;

replacing dimethylbenzene with paraffin, directly replacing dimethylbenzene with paraffin melted in advance in a thermostat at 60 ℃, repeating for 4 days for 1 time each day;

embedding a plant sample obtained after xylene is replaced by paraffin on a superclean platform treated by an RNA enzyme scavenger, placing a common enzyme-free square plastic culture dish serving as an embedding box on ice, adding about 1cm of clean paraffin melt liquid, quickly placing the sample in the space, removing bubbles around the plant sample by using a metal tweezers with burning flame, fully contacting the paraffin with the plant sample, fusing the paraffin into a whole, obtaining the embedded sample after the paraffin is solidified, and storing the embedded sample in a refrigerator at the temperature of-20 ℃.

On the basis of the technical scheme, in the step 2, the FAA fixative comprises: 30ml of absolute ethanol, 10ml of 37% formaldehyde, 5ml of glacial acetic acid and 55ml of RNase-free water.

On the basis of the technical scheme, in the step 3, the transcription template is obtained by directly synthesizing a DNA fragment by a company or amplifying the DNA fragment by using a high-fidelity PCR enzyme by using a primer containing a T7/Sp6 promoter sequence.

On the basis of the technical scheme, in the step 3, the probe purification specifically comprises the following steps: first, 75. mu.l of RNase-free water, 1. mu.l of 100mg/ml tRNA (Sigma, #10109517001) and 1. mu.l of DNase I (TaKaRa) were added to the in vitro transcription product after completion of the reaction in order to remove DNA by incubation at 37 ℃ for 30 min; then, the RNA product was recovered by absolute ethanol precipitation (equal volume of precooled 4M NH was added in sequence)₄Ac, 2 times volume of precooled absolute ethyl alcohol,slightly inverting and uniformly mixing, and placing in a refrigerator at the temperature of-20 ℃ for 3-6 hours; centrifuging at 14000rpm for 10min at 4 ℃ and discarding the supernatant to obtain a precipitate; rinsing the precipitate with 600 μ l 70% ethanol, centrifuging at 14000rpm for 7min at 4 deg.C, discarding the supernatant, sucking the supernatant instantly, and air drying the precipitate); adding 100 μ l RNase-free water for resuspension and precipitation, adding 100 μ l80mM sodium carbonate 120mM sodium bicarbonate buffer solution, maintaining at 60 deg.C for 50min for hydrolysis, and recovering RNA product by anhydrous ethanol precipitation; finally, 40 mul of 50% formamide is added to dissolve the precipitate to obtain the in-situ hybridization probe, and the in-situ hybridization probe is stored in a refrigerator at minus 80 ℃. The concentration of the in situ hybridization probe can be measured using Nanodrop or qubit3.0 before the in situ hybridization probe is placed in a-80 ℃ freezer.

On the basis of the above technical scheme, in step 5, the hybridization pretreatment process specifically comprises: placing the hybridization piece in a xylene solution 10min → a 50% xylene solution and a 50% ethanol solution 5min → a 100% ethanol solution 1min → a 95% ethanol solution 30s → an 85% ethanol solution 30s → a 70% ethanol solution 30s → a 50% ethanol solution 30s → a 0.85% NaCl solution 2min → a PBS buffer 2min → a proteinase K solution 37 ℃ reaction 30min → a 0.2% glycine solution 2min → a PBS buffer 2min → a 4% formaldehyde solution 10min → a PBS buffer2 acetic anhydride → a solution 10min → a PBS buffer 2min → a 0.85% NaCl solution 2min → a 50% ethanol solution 30s → a 70% ethanol solution 30s → an 85% ethanol solution 30s → a 95% ethanol solution 30s → a 100% ethanol solution 1min → 100% ethanol solution 1min, finishing hybridization pretreatment;

the proteinase K solution: prepared by RNase-free water, the substrate working concentration of the three solutions is 100mM Tris (pH8.0), 50mM EDTA, 1 ug/ml proteinase K (Sigma, # p2308), the dilution ratios are calculated according to the purchased related mother liquor and added in sequence, the solution is preheated at 37 ℃ in advance and later, and the proteinase K is added when in use;

the 0.2% glycine solution: preparing RNase-free PBS buffer solution, adding 0.2g of glycine into 100ml of RNase-free PBS buffer solution;

the 4% formaldehyde solution: preparing RNase-free water, a high-concentration PBS buffer solution and 37% formaldehyde, wherein the working concentration of a diluted solution substrate is 4% formaldehyde and 1 multiplied by PBS;

the acetic anhydride solution: preparing RNase-free water, wherein the working concentration of the solution substrate is 0.1M triethanolamine, 0.5% NaCl, 48mM HCl (prepared by concentrated hydrochloric acid), 0.025% acetic anhydride, adding acetic anhydride before use, and fully stirring and mixing.

On the basis of the above technical scheme, in step 6, the first hybridization incubation process is as follows:

firstly, moving the hybridization section sample obtained in the step 5 from a staining rack to a slide plate, and airing the hybridization section sample for 5-10min at room temperature until the hybridization section sample is pure white; meanwhile, the method comprises the steps of respectively diluting a sense probe and an antisense probe according to the consumption of 30 mul of diluted probe in each hybridization slice sample, wherein the 30 mul of diluted probe is the proportion of 2 mul of sense probe or antisense probe and 28 mul of 50% formamide solution; uniformly mixing the diluted sense probe or antisense probe, denaturing at 80 ℃ for 2min, immediately placing on ice for 2-3min, and instantaneously separating for 30 s;

then adding RNA hybridization solution after calculating according to 120 mul/tablet, sucking and uniformly mixing by using a cut gun head; then, 2 XSSC/50% formamide solution is added into the immunohistochemical wet box to cover the whole wet box bottom; then, 135 mul of hybridization mixed solution is added into each glass slide, after the liquid completely covers the sample, the glass cover is covered, and then the glass cover is placed into an immunohistochemical wet box; finally, after all the glass slides are placed in the immunohistochemical wet box, sealing the immunohistochemical wet box by using an adhesive tape, and placing the immunohistochemical wet box in a heat preservation box at 50-55 ℃ for overnight; the hybridization mixed solution is as follows: every 30. mu.l of diluted probe + 120. mu.l of RNA hybridization solution;

the RNA hybridization solution is as follows: preparing with RNase-free water, adding 500 μ l formamide (100%) for 1ml RNA hybridization solution, 200 μ l 50% dextran sulfate, 170 μ l RNase-free water, 100 μ l10 × Salts, 20 μ l50 × denhardt's, 10 μ l100mg/mltRNA as substrate dosage, preparing corresponding dosage according to the number of hybridization slices, wherein the dextran sulfate is viscous, the speed of pipetting is not too fast, and the solution is fully blown and evenly mixed;

the 10 × Salts solution is prepared from RNase-free water, and the working concentration of the solution substrate is 49mMNa₂HPO₄NaH at pH 6.8 at 51 mMm₂PO₄Buffer solution, 3M NaCl, 50M EDTA, and Tris/HCl pH8.0, and can be stored at-20 deg.C for a long period of time.

The 50% dextran sulfate: preparing with 60 deg.C RNase-free water, adding 0.5g dextran sulfate into 10ml RNase-free water, mixing well and dissolving, and storing at-20 deg.C for a long time;

the 2 XSSC/50% formamide: RNase-free water, 20 XSSC buffer and 100% formamide were used, and the working concentration of the diluted solution substrate was 2 XSSC and 50% formamide.

On the basis of the technical scheme, in the step 6, after hybridization incubation is carried out by using a primary antibody, a sample is processed before hybridization incubation is carried out by using a secondary antibody, and the specific processing process is as follows:

placing the sample subjected to hybridization incubation by using the primary antibody in a Buffer1 solution preheated at 55 ℃, removing a cover glass, and then reacting the Buffer1 solution at 55 ℃ for 1h → the Buffer1 solution at 55 ℃ for 1h → the Buffer2 solution at 37 ℃ for 5min → the Buffer2 solution at 37 ℃ for 5min → Buffer2+ RNaseA solution at 37 ℃ for 30min → the Buffer2 solution at 37 ℃ for 5min → the Buffer2 solution at 37 ℃ for 5min → the Buffer1 solution at 55 ℃ for 1h → TBS shaking table 5min → Buffer3 solution shaking table 1h → Buffer4 solution shaking table 45 min;

a second hybridization incubation was then performed using DIG-AP antibody solution: DIG-AP antibody hybridization incubation (each glass slide is washed by 100 mu l of a solution containing DIG-AP antibody, then sealed by 100 mu l of a DIG-AP antibody solution, put into a wet box added with Buffer4 for 2h at room temperature) → placing a clean staining rack after cover glass is removed from the Buffer4 solution → a Buffer4 solution shaking table 15min → a Buffer4 solution shaking table 15min → a Buffer4 solution shaking table 15min → a Buffer4 solution shaking table 15min → a Buffer5 solution shaking table 5min → a Buffer5 solution shaking table 5min, finally, in a dark environment, washing the sample slices once by 120 mu NBT/BCIP chromogenic substrate solution and pasting the two slides together, transferring into a common glass cup with 2ml NBT/BCIP chromogenic substrate Buffer at the bottom, wrapping and sealing the common glass cup by using a dark tin foil, placing the common glass cup in the dark environment, and carrying out chromogenic reaction at room temperature;

the Buffer1 solution: prepared according to the experimental dosage, 20 XSSC is diluted by distilled water to be 0.2 XSSC;

the Buffer2 solution: prepared by distilled water, three solutions of which the working concentration of the substrate is 0.5mM NaCl, 10mM pH7.5 Tris-HCl and 1mM EDTA are prepared according to the experimental dosage;

the Buffer3 solution: prepared by 60 ℃ 1 xTBS buffer solution, 100ml1 xTBS buffer solution is added with 1g skimmed milk powder (Roche, #11096176001), and the skimmed milk powder is continuously heated at 60 ℃ on a magnetic stirrer to ensure that the skimmed milk powder is fully dissolved;

the Buffer4 solution: prepared by TBS buffer solution and distilled water, the working concentration of a solution substrate is 1 xTBS, 1% BSA (Sigma, # sre0096) and 0.3% Triton X-100(Sigma, # t8787), and the solution substrate is prepared into proper dosage according to requirements and stirred on a magnetic stirrer at normal temperature to be fully and uniformly mixed;

the Buffer5 solution: prepared from distilled water, the working concentration of the solution substrate is 0.1mM Tris-HCl, 100mM NaCl and 50mM MgCl of pH 9.5₂The dosage is configured according to the requirement, and the components are fully and uniformly mixed;

the DIG-AP antibody solution: DIG-AP antibody (Roche, #11093274901) was diluted 1250-fold with Buffer4, i.e., 8. mu.l of DIG-AP antibody was added to 10ml of Buffer4 solution, and the total amount of preparation required was calculated based on the amount of 200. mu.l of AP antibody solution used per hybridization section sample.

On the basis of the technical scheme, in the step 6, the process of RNA hybridization liquid sealing is as follows: sucking RNA hybrid liquid containing an antibody by using a liquid transfer gun and dropping the RNA hybrid liquid on the left end of a hybrid slice sample, then lightly pressing the left end of the cover glass and pressing the left end with the left hand to ensure that the cover glass is attached with the RNA hybrid liquid, holding the other end of the cover glass by the right hand and slowly putting down, wherein the RNA hybrid liquid is slowly pushed away along with the cover glass, and the moving speed of the cover glass is required to be slower than that of the RNA hybrid liquid; in the RNA hybridization solution containing the antibody, the content of the primary antibody is 135 mul, and the content of the secondary antibody is 100 mul.

On the basis of the technical scheme, in the step 7, the NBT/BCIP chromogenic substrate buffer solution specifically comprises: the Buffer5 solution is used for preparation, and the working concentrations of two chromogenic substrates are as follows: 225. mu.g/ml NBT (Roche, #11383213001), 175. mu.g/ml BCIP (Roche, # 11383221001).

The invention provides an optimization aiming at the gene localization research of cucumber seedling mature tissues and insufficient application of the existing in-situ hybridization system. Firstly, according to the characteristics of cucumber seedling plant materials, improving the methods of plant culture, sampling and fixation, thereby obtaining a complete plant structure containing target tissues, which is the basis of the whole in-situ hybridization system; secondly, operation methods such as section preparation, hybridization pretreatment, hybridization incubation and sealing, chromogenic reaction microscopic examination and the like are optimized in a targeted manner, so that the reliability, accuracy and specificity of a gene positioning result are improved; finally, a treatment scheme suitable for the mature tissues of the cucumber seedlings after the termination of the chromogenic reaction is designed in a targeted manner, so that the interference of the cell contents is eliminated, and the distribution of the hybridization signals is directly observed.

Based on the three points, the invention constructs a complete in-situ hybridization system applied to mature tissues of cucumber seedlings, the optimization of the experimental technology is not only used for improving the sensitivity and accuracy of experimental results, but also the economic cost of the experimental system is reduced to a certain extent by methods of improving the utilization rate of medicines, reducing the consumption of experimental supplies, increasing the yield of reaction products, saving labor cost and the like, so the invention is a scientific research method which gives consideration to scientificity, innovativeness and practicability.

Drawings

The invention has the following drawings:

FIG. 1 is a comparison of a prior art in situ hybridization system with a prior art system of the present invention.

Wherein, A, the root system tissue of the cucumber seedling fixed by the existing in-situ hybridization technical system is shriveled, and the result is incomplete; B. the in-situ hybridization technical system is applied to complete root system tissue structure of cucumber seedlings and clear cell outline; C. the existing in situ hybridization technology system interferes the hybridization signal with the cell content; D. the in-situ hybridization technical system is applied to mature tissues of cucumber seedlings, and hybridization signals are clear and visible (white triangles point to the hybridization signals).

FIG. 2 is a schematic diagram of the sampling of root tissues of cucumber seedlings according to the present invention.

FIG. 3 is a schematic diagram of the design of a target gene probe according to the present invention.

Fig. 4 is a schematic diagram of the technical point of the spreading sheet of the present invention.

FIG. 5 is a schematic diagram of hybridization pretreatment of the in situ hybridization technique system of the present invention.

FIG. 6 is a schematic diagram of the hybridization incubation mounting plate of the present invention.

FIG. 7 is a schematic diagram of the hybridization post-treatment of the in situ hybridization system of the present invention.

FIG. 8 is a diagram showing the result of the location of CsNPF7.2 gene in cucumber caulicles.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings.

1 cultivation of test Material

The overground tissue material of cucumber seedlings has no special requirements on culture conditions, the quality of the underground tissue material is related to the culture mode, and the tissue culture, water culture, sand culture and soil culture (fine soil) can not cause the epidermis of the root system of the seedling to adhere to impurities which are difficult to remove, so the method is a recommended culture method.

2 sampling and fixing of test Material

Sampling on ice, wherein the stem lignification degree of the cucumber seedling is low, the root is fine and soft, the tissue sampling is carried out, the stem with the length of about 3-4 cm and the root segment containing the lateral root are cut, and as shown in figure 2, the embedded sample is cut into smaller tissue samples when the embedded sample is subjected to block repairing before slicing;

the taken plant material is quickly put into precooled FAA stationary liquid (improved formula: 30ml of absolute ethyl alcohol, 10ml of 37% formaldehyde, 5ml of glacial acetic acid and 55ml of RNase-free water), vacuum pumping is carried out by a vacuum pump for about 18-20 min, then air is slowly introduced to restore atmospheric pressure, new FAA stationary liquid is replaced and shaken on a horizontal shaking table at 4 ℃ for overnight, and the sample can be stored for 1 week in a short time at 4 ℃.

Samples were dehydrated on a horizontal shaker at 4 ℃ with ethanol concentration gradients of 50%, 70%, 85%, 95%, 100% (formulated from RNase-free water), from low to high concentration, each gradient being supplemented with a small amount of eosin stain (used to distinguish paraffin from sample during sectioning) until the plant sample was stained pink, the dehydration time for each gradient was modified to around 35min, and the last 100% ethanol gradient was overnight.

The xylene replacing ethanol is horizontally shaken at normal temperature, and sequentially comprises 100% ethanol, 50% ethanol/50% xylene, 100% xylene and 100% xylene, the replacing time of each solution is about 30min, and the last 100% xylene is overnight.

Paraffin-substituted xylene the xylene was directly substituted with previously melted clean paraffin in a 60 ℃ incubator for 4 days 1 time per day.

The sample is embedded in a super clean bench treated by an RNase scavenger, a common enzyme-free square plastic culture dish is used as an embedding box and placed on ice, about 1cm of clean paraffin melting liquid is added, then the sample is rapidly placed in the super clean bench, then bubbles around the sample are removed by a metal forceps which is scorching by flame, the paraffin is fully contacted with the sample and is melted into a whole, and the paraffin is placed in a refrigerator at the temperature of-20 ℃ for storage after being solidified.

3 Probe preparation of target Gene

As shown in FIG. 3, the transcription template of the probe consists of a template fragment and a T7/Sp6 promoter sequence, the connection direction of a sense probe and an antisense probe is opposite to that of the promoter, the sense probe is the negative control of a target gene, the antisense probe is a complementary sequence of the target gene, the antisense probe is combined with the target gene to display the position of the target gene, and the antisense probe and the target gene are transcription templates in vitro transcription products with the target gene. According to the mRNA sequence length of a target gene and a fluorescence quantitative qPCR result, a target gene fragment with GG initiation and GC content of 40% -70% is selected as a template fragment, and the longer the mRNA sequence length is, the higher the fluorescence quantitative delta CQ value is, and the longer the template fragment is. The length of the template fragment of mRNA of about 2000bp is generally 500 bp-1500 bp.

The transcription template can be prepared by synthesizing DNA fragments directly from the company, or by amplifying the DNA fragments with high-fidelity PCR enzyme using primers containing the T7/Sp6 promoter sequence (FIG. 3).

The in vitro transcription system does not need to require the total amount of template DNA to be more than 1 mu g, partial RNA products can be lost in the purification stage of in vitro transcription products, the time for ethanol or isopropanol precipitation at the temperature of 20 ℃ below zero is increased to be more than 3h (can be over night), and the yield of the probe can be increased. About 150ng of transcription template is initially used, and the transcription product can be recovered to about 100 mu g in reaction time of 2 hours.

4 preparation of hybrid sections

Hybridization signals of mature tissues are different from those of meristems, signal molecules are scattered in single cells, tissue sections of samples are not too thin, and the number of target molecules fixed on the tissues is reduced. Mature tissue slices of 10-12 mu m thick cucumber seedlings are suitable for in-situ hybridization experiments.

The paraffin sections prepared by the slicing machine are screened and then spread, the paraffin sections which are connected into a strip are cut into the length suitable for placing the glass slide by a blade, the paraffin sections are gently placed on the glass slide, the paraffin sections are placed under a microscope for simple microscopic examination and screening, and the sections with incomplete structures, such as tissue fragmentation, cavities, blade scratches and the like, are discarded.

The screened sections are gently placed in RNase-free water at 37 ℃ for spreading, as shown in figure 4, the sections with paraffin are softened and then are completely spread, the color is changed from white to almost transparent, the sections are gently dragged and spread on the glass slide by a writing brush, bubbles are removed by the light pressure of the writing brush, and water is removed by the light pressure of water absorption paper, so that the time for drying the sections is shortened, the effect of drying the sections is improved, and the separation of a later-period sample from the glass slide is avoided.

In the slicing process, wax stains on the blade are checked and cleaned in time, and sample fragmentation caused by scratches is avoided.

5 hybridization pretreatment

Hybridization pretreatment Paraffin-coated tissue samples were dewaxed, destained, and the proteins coating the target molecules were treated to expose the target molecules sufficiently, so all the tools should be treated with RNase scavenger, all reagents being formulated with RNase-free water.

The process is optimized by using PBS buffer solution, gradient ethanol and the like for multiple times, as shown in figure 5, and finally, the sample is stored in a refrigerator at 4 ℃ and placed on a staining rack in a staining jar sealed by a sealing film with a small amount of absolute ethanol added at the bottom.

6 hybrid incubation mounting

In the in situ hybridization system, a primary antibody and a secondary antibody are respectively hybridized and incubated, an RNA hybridization solution containing the antibody (the primary antibody is a DIG-11-UTP probe, and the secondary antibody is DIG-AP) is used for rinsing a tissue sample, and then an RNA hybridization solution mounting sheet is used for incubation, so that the hybridization effect is determined intuitively by the degree of the RNA hybridization solution mounting.

The RNA hybridization solution sealing, i.e. sample sealing, requires that the tissue sample must be completely soaked in the RNA hybridization solution, and once bubbles are generated, the sample at the position is not contacted with the antibody, thereby affecting the accuracy of the gene positioning result.

The optimized mounting technology is shown in figure 6, a pipette sucks a proper amount (primary antibody-135 mul, secondary antibody-100 mul) of RNA hybridization liquid to be dropped at the position shown in figure 6, then one side of a cover glass is slightly pressed down at the side and is held down by the left hand to enable the cover glass to be attached with the RNA hybridization liquid, the other layer of the cover glass is slowly put down by the right hand, the RNA hybridization liquid is slowly pushed away along with the cover glass, and the moving speed of the cover glass is required to be slower than that of the RNA hybridization liquid, particularly the primary anti-RNA hybridization liquid which is thickened by adding dextran polysulfate.

7 color reaction microscopic examination

The in-situ hybridization system of the mature tissue of the cucumber seedling is not suitable for direct termination of the chromogenic reaction after three days, and in order to reduce background noise as much as possible and improve the specificity of target gene positioning, a hybridization signal molecule should be discovered as early as possible, and the possibility that the signal appearing earlier is noise is lower.

The gene with high relative expression abundance of the gene in a special period, namely the gene with CQ value of 15-25 of the fluorescence quantitative PCR and CQ value of the internal reference gene of 15, should be checked for the hybridization signal by performing a microscopic examination within 6h after the color reaction begins; genes with general expression abundance, namely genes with CQ value of more than or equal to 25 and less than or equal to 30 of fluorescence quantitative PCR and CQ value of internal reference genes of 15, are subjected to microscopic examination 2 times each day in the morning, afternoon or evening from the next day.

Screening light as far as possible during microscopic examination, separating the hybridization slice sample after mounting in a chromogenic substrate Buffer solution (Buffer5), preparing a temporary mounting by using the Buffer solution, then placing under a microscope to examine a hybridization signal, and stopping the chromogenic reaction if a clear hybridization signal (purple red) molecule is observed; otherwise, the sample was rinsed with the chromogenic reaction solution and the reaction was continued by returning the gel to the Colophony glass.

8 post-treatment after termination of the reaction

The cells of the mature tissue of cucumber seedlings contain abundant inclusion impurities, which are hardly soluble in water, ethanol and xylene, and microscopically black impurities seriously interfere with the hybridization signal (fig. 1). For tissue samples where hybridization signals were observed, the color reaction was stopped with clear water, then left to air dry at room temperature, and finally each slide was directly coated with 90 μ l of neutral resin: and sealing the slices by using mixed liquor with the ratio of dimethylbenzene to dimethylbenzene being 1: 1. The sample after the mounting is hardly influenced by impurities in the content, the hybridization signal is clear, and the experimental result is permanently stored.

(to aid in further understanding of the technical solution); the invention is illustrated by the specific examples;

example (b): CsNPF7.2 gene in situ hybridization of cucumber caulicles

1 test Material

Tissue culture: selecting 'Xintai Mici' cucumber seeds with consistent size, shape and weight, soaking the seeds in distilled water at 55-60 ℃ for about 30min, and then peeling the seed coats of the cucumbers by using small tweezers (the tweezers are prevented from contacting the growing points of the seeds as much as possible); sterilizing with 75% alcohol for 30s, cleaning with sterilized water for 3 times, sterilizing with 2.5% sodium hypochlorite for 10min, cleaning with sterilized water for 3 times, placing the treated seeds on MS solid culture medium, transferring into plant incubator of south of the Yangtze river, and setting photoperiod as follows: the first stage is dark culture at 28 ℃ for 24 h; the second stage has photoperiod of 12h, 28 deg.C in the daytime and 18 deg.C in the evening, and is cultured for 13 days.

2 sampling and fixing of test Material

Soaking the cucumber caulicles which are vacuumized by FAA fixing solution, then respectively carrying out ethanol gradient dehydration, xylene replacement ethanol and paraffin replacement xylene, finally embedding a sample by paraffin and storing the sample at-20 ℃.

3 Probe preparation of target Gene

A transcription template amplification primer of CsNPF7.2 is designed by utilizing SnapGene 4.1.8 software as shown in Table 1, a high-fidelity PCR enzyme PrimeSTAR (TaKaRa) amplification product is recovered from glue and is used as a cDNA of the CsNPF7.2 to obtain a transcription template, then an in vitro transcription reaction system containing Digoxigenin-11-UTP (biotin digoxin-labeled uracil nucleotide) is configured according to the guidance of a purchased T7/Sp6 in vitro transcription instruction, and the reaction is carried out for 2h at 37 ℃.

TABLE 1 CsNPF7.2 Gene in situ hybridization Probe primers

Note: the sense probe is connected with the T7 promoter, the antisense probe is connected with the Sp6 promoter, and the sequence marked with the horizontal line in the table is the promoter sequence.

And (3) probe purification: firstly, 75 μ l of RNase-free water, 1 μ l of 100mg/ml tRNA (Sigma, #10109517001) and 1 μ l of DNase I (TaKaRa) are sequentially added into in vitro transcription products after the reaction is finished, and the temperature is kept at 37 ℃ for 30min to remove DNA; then, the RNA product was recovered by absolute ethanol precipitation (equal volume of precooled 4M NH was added in sequence)₄Ac, 2 times of volume of precooled absolute ethyl alcohol, slightly reversing and uniformly mixing, and placing in a refrigerator at the temperature of minus 20 ℃ for 3-6 hours; centrifuging at 14000rpm at 4 ℃ for 10min, and discarding the supernatant; rinsing the precipitate with 600 μ l 70% ethanol, centrifuging at 14000rpm at 4 deg.C for 7min, discarding the supernatant, instantly sucking the supernatant, and air drying the precipitate); adding 100 μ l RNase-free water for resuspension and precipitation, adding 100 μ l80mM sodium carbonate 120mM sodium bicarbonate buffer solution, maintaining at 60 deg.C for 50min for hydrolysis, and recovering RNA product by alcohol precipitation; finally, 40. mu.l of 50% formamide was added to dissolve the precipitate and the in situ hybridization probe was stored in a freezer at-80 ℃. The concentration of the RNA probe can be measured using a Nanodrop or a Qubit3.0 before the probe is placed in an ultra-low temperature refrigerator.

4 preparation of hybridization samples

Block repairing: igniting an alcohol lamp, taking a clean newspaper and placing the newspaper on a table, and placing a 2ml centrifuge tube rack and a 2ml centrifuge tube; the successfully embedded sample is then removed and cut with a surgical blade into a sample-containing wax block of about 0.5mm by 0.5mm in volume around the sample (cut off excess paraffin, the closer the edge is to the sample the better); putting a small piece of broken paraffin on a centrifuge tube cover, using a blade burnt by alcohol flame to melt the paraffin, then quickly putting the cut paraffin, and fixing the paraffin on the centrifuge tube along with the solidification of the paraffin; marking the name of the sample on the centrifugal tube by using a sign pen;

slicing: opening the slicer, returning the sample loading head to the original position, and setting the slice thickness to be 10 mu m; then, a sample with a successful trimming block is placed, and the position of a blade is adjusted to be right aligned with the sample to form a tangent plane; then, rotating the slicer, and pushing out the paraffin sections formed into strips with the assistance of a writing brush to prevent the samples from being stuck together and forming lumps;

spreading: hooking a next section of sample by using a fine hair pen, putting the next section of sample on clean A4 paper, cutting the sample into small sections by using a blade, putting the small sections on a glass slide, and preliminarily screening out a tissue which is complete, free of damage and is a desired tissue structure by using a microscope; carefully placing the selected small fragments into a 37 ℃ water bath flaking machine, and carefully flattening the sample; finally, the sample is fished up by the glass slide to be stuck on the glass slide, the water absorption paper carefully absorbs the redundant water from the periphery of the sample, and the sample is pushed by the brush pen to remove the bubbles;

baking the slices: and (3) putting the successfully-spread glass slide on a 42 ℃ wafer drier overnight, so that the sample is completely stuck on the glass slide, and the dewaxed tissue cannot fall off.

5 hybridization pretreatment process:

the experiments were carried out: in the stage, reagents required are prepared from RNase-free water, except common reagents of dimethylbenzene, gradient ethanol (100-95-85-70-50%), 0.85% NaCl, EDTA and PBS buffer solution, related solutions of protease K solution, 0.2% glycine solution, 4% formaldehyde solution, acetic anhydride solution and the like are prepared according to the required dosage of an experiment before the start of the experiment, and the formulas are as follows:

protease K solution: RNase-free water was prepared, the substrate working concentrations of the three solutions were 100mM Tris (pH8.0), 50mM EDTA, and 1. mu.g/ml proteinase K (Sigma, # p2308), and the solutions were sequentially added at dilution ratios calculated from the respective stock solutions purchased, preheated at 37 ℃ one night in advance, and proteinase K was added when used.

0.2% glycine solution: RNase-freePBS buffer, 0.2g of glycine was added to 100ml of the buffer.

4% formaldehyde solution: RNase-free water, high-concentration PBS buffer solution and 37% formaldehyde are prepared, and the working concentration of the diluted solution substrate is 4% formaldehyde and 1 XPBS.

Acetic anhydride solution: preparing RNase-free water, wherein the working concentration of the solution substrate is 0.1M triethanolamine, 0.5% NaCl, 48mM HCl (prepared by concentrated hydrochloric acid), 0.025% acetic anhydride, adding acetic anhydride before use, and fully stirring and mixing.

6 first hybridization incubation (first antibody)

Probe dilution: the amount of the probe diluted was calculated in 30. mu.l per section (2. mu.l of the sense probe or antisense probe + 28. mu.l of 50% formamide solution), and the sense probe and the antisense probe were diluted separately.

Firstly, moving a glass slide from a staining rack to a glass slide plate, and airing the glass slide for 5-10min at room temperature until a tissue sample is pure white; meanwhile, the diluted probe was denatured at 80 ℃ for 2min, immediately placed on ice for 2-3min, and instantaneously dissociated for 30 s. Then adding corresponding RNA hybridization solution after calculating according to 120 mul/tablet, sucking and stirring uniformly by using a cut gun head; then, adding a proper amount of 2 XSSC/50% formamide solution into the immunohistochemical wet box; then, 135 mul of hybridization mixed solution is added into each glass slide, after the liquid completely covers the sample, the glass cover is covered, and then the glass cover is placed into a wet box; and finally, after all the glass slides are placed in a wet box, sealing the wet box by using an adhesive tape, and placing the wet box in an incubator at 50-55 ℃ for overnight.

The reagents required in the stage are also prepared from RNase-free water, common reagents of formamide, tRNA, SSC buffer solution and 50 XDenhardt's can be directly purchased and used, and before the test is started, related solutions of 10 XSalts, 50% dextran sulfate, RNA hybridization solution, 2 XSSC/50% formamide and the like are required to be prepared in situ, and the formula is as follows:

10 × Salts solution: the RNase-free water is prepared, the working concentration of the solution substrate is 49mMNa2HPO4 & 51mMNaH2PO4 buffer solution (pH 6.8), 3MNaCl, 50mMEDTA and Tris & HCl (pH8.0), and the solution can be stored for a long time at the temperature of minus 20 ℃.

50% dextran sulfate: prepared with RNase-free water heated at 60 deg.C, 0.5g dextran sulfate is added into 10ml RNase-free water, and the mixture is well mixed and dissolved, and can be stored at-20 deg.C for a long time.

RNA hybridization solution: preparing RNase-free water, wherein the dosage of the RNA hybridization solution of each slice is 120 mu l, the dosage of the substrate added into 1ml of RNA hybridization solution is 500 mu l of formamide (100%), 200 mu l of 50% dextran sulfate, 170 mu l of RNase-free water, 100 mu l of 10 xsalts, 20 mu l of 50 xdenhardt's and 10 mu l of 100mg/mltRNA, and the corresponding dosage is prepared according to the number of the hybridization slices, wherein the dextran sulfate is viscous, the pipetting speed of a pipetting gun is not too fast, and the pipetting is fully and uniformly blown.

2 × SSC/50% formamide: RNase-free water, 20 XSSC buffer and 100% formamide were used, and the working concentration of the diluted solution substrate was 2 XSSC and 50% formamide.

7 second hybridization incubation (second antibody)

As shown in FIG. 7, the sample was treated before the second hybridization incubation, and then each slide was washed with 100. mu.l of the DIG-AP antibody-containing solution, then mounted with 100. mu.l of the DIG-AP antibody solution, and incubated in a wet box with Buffer4 for 2h at room temperature;

after the second hybridization incubation was completed, the cover slip was washed off, and then washed 4 times with Buffer4 for 15min each and 2 times with Buffer5 for 5min each, as shown in fig. 7.

Finally, in a dark environment, washing the sample slice once with 120 μ l of NBT/BCIP chromogenic substrate solution, pasting the glass slides together, transferring into a Cooprine cup with the bottom added with 2ml of NBT/BCIP chromogenic substrate solution, wrapping and sealing with tin foil paper, placing in a dark environment, and carrying out chromogenic reaction at room temperature.

In addition to the commonly used reagents, the experiment at this stage needs to purchase 10 xTBS Buffer solution, RNaseA, BSA (bovine serum albumin), skimmed milk powder (Roche), Triton X-100(Sigma), DIG-AP (Roche) and chromogenic substrate NBT/BCIP (Roche), before the experiment is started, five Buffer solutions, namely Buffer 1(0.2 xSSC), Buffer 2(NTE), Buffer3, Buffer4, Buffer5, two reaction solutions, namely DIG-AP antibody solution and NBT/BCIP display substrate solution, are prepared in advance, and the solution at this stage is prepared by common distilled water, and the formula of the solution is as follows:

buffer1 solution: according to the experimental amount, 20 XSSC is diluted to 0.2 XSSC by distilled water.

Buffer2 solution: prepared from distilled water, the working concentration of the three solutions of substrate is 0.5mM NaCl, 10mM Tris & HCl (pH 7.5) and 1mM EDTA, and the three solutions are prepared according to the experimental dosage.

Buffer3 solution: prepared from 60 ℃ 1 × TBS buffer, 100ml1 × TBS buffer was added with 1g skim milk powder (Roche, #11096176001), and heated continuously on a magnetic stirrer at 60 ℃ to ensure that the skim milk powder was well dissolved.

Buffer4 solution: prepared from TBS buffer solution and distilled water, the working concentration of the solution substrate is 1 xTBS, 1% BSA (Sigma, # sre0096) and 0.3% Triton X-100(Sigma, # t8787), and the solution substrate is prepared into proper dosage according to requirements, and stirred on a magnetic stirrer at normal temperature to be fully mixed.

Buffer5 solution: prepared from distilled water, the working concentration of the solution substrate was 0.1M Tris & HCl (pH 9.5), 100mM NaCl, 50mM MgCl₂The dosage is configured according to the requirement and the mixture is fully and evenly mixed.

DIG-AP antibody solution: DIG-AP antibody (Roche, #11093274901) was diluted 1250-fold with Buffer4, i.e., 8. mu.l of DIG-AP antibody was added to 10ml of Buffer4 solution, and the total amount of preparation required was calculated based on the amount of 200. mu.l of AP antibody solution per slide.

NBT/BCIP shows substrate solution: prepared by using Buffer5 solution, the working concentration of two chromogenic substrates is as follows: 225. mu.g/ml NBT (Roche, #11383213001), 175. mu.g/ml BCIP (Roche, #11383221001), according to the configuration dosage required by the experiment.

8, color development treatment process after hybridization:

In a special period, the gene with high relative expression abundance should be examined by microscopic examination within 6h from the beginning of the color reaction to check the hybridization signal, and the general genes are examined by microscopic examination 2 times a day in the morning, afternoon or evening every day.

During microscopic examination, keeping the slide glass away from light as much as possible, separating the adhered slide glass in an NBT/BCIP chromogenic substrate buffer solution, making a temporary loading piece by using the NBT/BCIP chromogenic substrate buffer solution, then placing the temporary loading piece under a microscope to examine a hybridization signal, and if clear hybridization signal (purple red) molecules are observed, terminating the chromogenic reaction; otherwise, the sample was rinsed again with NBT/BCIP chromogenic substrate buffer, and the reaction was continued by returning the sticks to the Cooprin cup.

9 color reaction termination and mounting:

the cells of the mature tissue of cucumber seedlings contain abundant inclusion impurities which are hardly soluble in water, ethanol and xylene, and impurities like black under a microscope seriously interfere with hybridization signals. For tissue samples where hybridization signals were observed, the color reaction was stopped with clear water, then left to air dry at room temperature, and finally each slide was directly coated with 90 μ l of neutral resin: and sealing the slices by using mixed liquor with the ratio of dimethylbenzene to dimethylbenzene being 1: 1. The sample after the mounting is hardly influenced by impurities in the content, the hybridization signal is clear, and the experimental result is permanently stored. The positioning result of CsNPF7.2 in cucumber caulicles is shown in figure 8.

Those not described in detail in this specification are within the skill of the art.

Reference to the literature

Huang,Sanwen et al.2009.“The Genome of the Cucumber,Cucumis SativusL.”Nature genetics41(12):1275.

“In Situ Hybridization Protocol(for Plant Roots)–Benfey Lab.”2019.

https://sites.duke.edu/benfey/protocols/in-situ-hybridization-protocol-for-plant-roots/(June 5,2019).

JACKSON,and DAVID.1991.“In Situ Hybridization in Plants.”In MolecularPlant Pathology:A Practical Approach,Oxford University Press.

Meyerowitz,and Elliot M.1987.“In Situ Hybridization to RNA in PlantTissue.”Plant Molecular Biology Reporter 5(1):242–50.

Chenke, bright-red, 37154;, Wenhao, and Europe also Laozi, 2018, "RNA in situ hybridization," Bio-protocol: e1010119.https:// doi.org/10.21769/BioProtoc.1010119.

Claims

1. An RNA in-situ hybridization optimization method for cucumber seedling gene localization research is characterized by comprising the following steps:

step 2, sampling and fixing a test material;

sampling on ice, cutting stems of cucumber seedlings with the length of 3-4 cm and root segments containing lateral roots as plant samples, embedding to obtain embedded samples, and trimming and cutting the embedded samples to obtain tissue samples;

step 3, preparing a probe of the target gene, and purifying the probe;

step 4, preparing a hybridization slice;

the screened paraffin sections are flatly placed in RNase-free water at 37 ℃ for spreading, the paraffin sections are fully spread after being softened, the color is changed from white to almost transparent, the paraffin sections are lightly dragged and spread on a glass slide by a writing brush, bubbles are removed by the light pressure of the writing brush, and water is absorbed by the light pressure of absorbent paper, so that the time for drying the paraffin sections is shortened, the effect of drying the paraffin sections is improved, the later-stage separation of samples from the glass slide is avoided, and the hybrid sections are obtained;

step 5, hybridization pretreatment;

step 6, hybridization incubation mounting:

step 7, microscopic examination of color reaction:

when the CQ value of the fluorescent quantitative PCR of the cucumber seedling stage gene is more than or equal to 15 and less than 25 and the CQ value of the internal reference gene is 15, performing microscopic examination once within 6 hours after the color reaction starts to check a hybridization signal; when the CQ value of the fluorescent quantitative PCR of the cucumber seedling stage gene is more than or equal to 25 and less than or equal to 30 and the CQ value of the internal reference gene is 15, performing microscopic examination 2 times a day in the morning, afternoon or evening respectively on the next day;

placing the hybridization slice sample subjected to the sealing in the step 6 in NBT/BCIP chromogenic substrate buffer solution for separation in light shielding during microscopic examination, making a temporary mounting by using the NBT/BCIP chromogenic substrate buffer solution, then placing the temporary mounting under a microscope for detecting a hybridization signal, and stopping the chromogenic reaction if clear hybridization signal molecules are observed; otherwise, the sample is washed again by NBT/BCIP chromogenic substrate buffer solution, and then the two glass slides are bonded again and put back into the Cooprin cup for continuous reaction;

and 8, reaction termination post-treatment:

for tissue samples where hybridization signals were observed, the color reaction was stopped with clear water, then left to air dry at room temperature, and finally each slide was directly coated with 90 μ l of neutral resin: sealing the mixed solution with the ratio of 1:1 of dimethylbenzene, and permanently storing experimental results;

in step 6, the first hybridization incubation process is as follows:

the RNA hybridization solution is as follows: preparing RNase-free water, adding 500 ul 100% formamide, 200 ul 50% dextran sulfate, 170 ul RNase-free water, 100 ul 10 xsalts, 20 ul 50 xdenhardt's and 10 ul 100mg/ml tRNA as substrate for 1ml RNA hybridization solution, preparing corresponding dosage according to the number of the hybridization slices, wherein the dextran sulfate is viscous, the pipetting speed of a pipetting gun is not too fast, and the solution is fully and evenly blown;

the 10 × Salts solution is prepared from RNase-free water, and the working concentration of the solution substrate is 49mM Na₂HPO₄NaH at pH 6.8 at 51 mMm₂PO₄Buffer solution, 3M NaCl, 50mM EDTA, Tris & HCl with pH8.0, can be stored for a long time at-20 ℃;

the 50% dextran sulfate: preparing with 60 deg.C RNase-free water, adding 0.5g dextran sulfate into 10ml RNase-free water, mixing well, dissolving, and storing at-20 deg.C for a long time;

the 2 XSSC/50% formamide: preparing RNase-free water, 20 XSSC buffer solution and 100% formamide, wherein the working concentration of a diluted solution substrate is 2 XSSC and 50% formamide;

in step 6, after hybridization incubation with the primary antibody and before hybridization incubation with the secondary antibody, the sample is processed, specifically, the processing process is as follows:

a second hybridization incubation was then performed using DIG-AP antibody solution: DIG-AP antibody hybridization incubation → after cover glass is removed from the Buffer4 solution, placing a clean staining rack → Buffer4 solution shaking table 15min → Buffer4 solution shaking table 15min → Buffer4 solution shaking table 15min → Buffer4 solution shaking table 15min → Buffer5 solution shaking table 5min → Buffer5 solution shaking table 5min, finally, in a dark environment, washing the sample slices once with 120 μ l NBT/BCIP chromogenic substrate solution and pasting the glass slides together in pairs, transferring to a Cooprin cup with 2ml NBT/BCIP chromogenic substrate Buffer solution added at the bottom, wrapping and sealing with tin foil, placing in a dark environment, and carrying out chromogenic reaction at room temperature;

the DIG-AP hybridization incubation specifically comprises the following steps: washing each glass slide by using 100 mu l of DIG-AP antibody-containing solution, then sealing by using 100 mu l of DIG-AP antibody solution, and putting the glass slide into a wet box with Buffer4 to incubate for 2 hours at room temperature;

the Buffer2 solution: prepared by distilled water, the working concentration of three solution substrates is 0.5mM NaCl, 10mM Tris-HCl with pH7.5 and 1mM EDTA, and the three solution substrates are prepared according to the experimental dosage;

the Buffer3 solution: prepared by 60 ℃ 1 xTBS buffer solution, 100ml1 xTBS buffer solution is added with 1g of skimmed milk powder, and the skimmed milk powder is continuously heated at 60 ℃ on a magnetic stirrer to ensure that the skimmed milk powder is fully dissolved;

the Buffer4 solution: the TBS solution is prepared by TBS buffer solution and distilled water, the working concentration of a solution substrate is 1 xTBS, 1% BSA and 0.3% Triton X-100, and the appropriate dosage is prepared according to requirements, and the solution substrate is stirred on a magnetic stirrer at normal temperature to be fully and uniformly mixed;

the DIG-AP antibody solution: DIG-AP antibody was diluted 1250-fold with Buffer4, i.e., 8. mu.l of DIG-AP antibody was added to 10ml of Buffer4 solution, and the total amount of preparation required was calculated based on the amount of 200. mu.l of DIG-AP antibody solution used per sample of hybridization section.

2. The RNA in situ hybridization optimization method for cucumber seedling gene mapping research as claimed in claim 1, wherein in step 2, the embedding process is as follows:

embedding the plant sample obtained after xylene is replaced by paraffin on a superclean platform treated by an RNA enzyme scavenger, placing a common enzyme-free square plastic culture dish serving as an embedding box on ice, adding paraffin melting liquid with the thickness of 1cm, rapidly placing the plant sample in the plant sample, removing bubbles around the plant sample by using a metal tweezers with burning flame, fully contacting and melting the paraffin and the plant sample into a whole, obtaining the embedded sample after the paraffin is solidified, and storing the embedded sample in a refrigerator at the temperature of-20 ℃.

3. The RNA in situ hybridization optimization method for cucumber seedling gene location research as claimed in claim 2, wherein in step 2, the FAA fixative comprises: 30ml of absolute ethanol, 10ml of 37% formaldehyde, 5ml of glacial acetic acid and 55ml of RNase-free water.

4. The RNA in situ hybridization optimization method for cucumber seedling gene location research as claimed in claim 1, wherein, in step 3, the transcription template is obtained by direct synthesis of DNA fragments by a company or by amplification with high fidelity PCR enzyme by using primers containing T7/Sp6 promoter sequences.

5. The RNA in situ hybridization optimization method for cucumber seedling gene mapping research as claimed in claim 1, wherein in step 3, the probe purification specifically comprises: firstly, adding 75 mu l of RNase-free water, 1 mu l of tRNA 100mg/ml and 1 mu l of DNase I into in-vitro transcription products after reaction is finished in sequence, and preserving the temperature at 37 ℃ for 30min for removing DNA; then, the RNA product is recovered by absolute ethyl alcohol precipitation method, and equal volume of precooled 4M NH is added in sequence₄Ac, 2 times of volume of precooled absolute ethyl alcohol, slightly reversing and uniformly mixing, and placing in a refrigerator at the temperature of minus 20 ℃ for 3-6 hours; centrifuging at 14000rpm for 10min at 4 ℃ and discarding the supernatant to obtain a precipitate; 600. mu.l of 70 for precipitationRinsing with% ethanol, centrifuging at 14000rpm for 7min at 4 deg.C, discarding the supernatant, instantly sucking the supernatant, and waiting for precipitation and air drying; adding 100 μ l RNase-free water for resuspension and precipitation, adding 100 μ l80mM sodium carbonate 120mM sodium bicarbonate buffer solution, maintaining at 60 deg.C for 50min for hydrolysis, and recovering RNA product by anhydrous ethanol precipitation; finally, 40 mul of 50 percent formamide is added to dissolve the precipitate to obtain the in-situ hybridization probe, and the in-situ hybridization probe is stored in a refrigerator at the temperature of minus 80 ℃; the concentration of the in situ hybridization probe was measured using Nanodrop or qubit3.0 before the in situ hybridization probe was placed in a-80 ℃ freezer.

6. The RNA in-situ hybridization optimization method for cucumber seedling gene mapping research as claimed in claim 1, wherein in step 5, the hybridization pretreatment process specifically comprises the following steps: placing the hybridization piece in a xylene solution 10min → a 50% xylene solution and a 50% ethanol solution 5min → a 100% ethanol solution 1min → a 95% ethanol solution 30s → an 85% ethanol solution 30s → a 70% ethanol solution 30s → a 50% ethanol solution 30s → a 0.85% NaCl solution 2min → a PBS buffer 2min → a proteinase K solution 37 ℃ reaction 30min → a 0.2% glycine solution 2min → a PBS buffer 2min → a 4% formaldehyde solution 10min → a PBS buffer2 acetic anhydride → a solution 10min → a PBS buffer 2min → a 0.85% NaCl solution 2min → a 50% ethanol solution 30s → a 70% ethanol solution 30s → an 85% ethanol solution 30s → a 95% ethanol solution 30s → a 100% ethanol solution 1min → 100% ethanol solution 1min, finishing hybridization pretreatment;

the proteinase K solution: prepared by RNase-free water, the working concentrations of three solutions of substrates are respectively as follows: 100mM Tris with pH of 8.0, 50mM EDTA, 1 μ g/ml proteinaseK, calculating dilution ratio according to the purchased related mother liquor, sequentially adding, preheating at 37 ℃ one night in advance, and adding the proteinaseK when in use;

the acetic anhydride solution: preparing RNase-free water, adding acetic anhydride before use, and mixing thoroughly, wherein the working concentration of the solution substrate is 0.1M triethanolamine, 0.5% NaCl, 48mM HCl and 0.025% acetic anhydride.

7. The RNA in situ hybridization optimization method for cucumber seedling gene mapping research as claimed in claim 1, wherein in step 6, the process of RNA hybridization liquid sealing is as follows: sucking RNA hybrid liquid containing an antibody by using a liquid transfer gun and dropping the RNA hybrid liquid on the left end of a hybrid slice sample, then lightly pressing the left end of the cover glass and pressing the left end with the left hand to ensure that the cover glass is attached with the RNA hybrid liquid, holding the other end of the cover glass by the right hand and slowly putting down, wherein the RNA hybrid liquid is slowly pushed away along with the cover glass, and the moving speed of the cover glass is required to be slower than that of the RNA hybrid liquid; in the RNA hybridization solution containing the antibody, the content of the primary antibody is 135 mul, and the content of the secondary antibody is 100 mul.

8. The RNA in-situ hybridization optimization method for cucumber seedling gene mapping research as claimed in claim 1, wherein the NBT/BCIP chromogenic substrate buffer solution is specifically: the Buffer5 solution is used for preparation, and the working concentrations of two chromogenic substrates are as follows: 225. mu.g/ml NBT, 175. mu.g/ml BCIP.