CN104975078A - Method for detecting target siRNA pool in transgene plant - Google Patents

Abstract

The invention discloses a method for detecting a target siRNA pool in a transgenic plant, which comprises: using the target gene DNA as a template to prepare a DIG-UTP-labeled antisense RNA chain: the reaction system includes: UTP, ATP, GTP, CTP, DIG- UTP, RNA polymerase buffer, target DNA, RNA polymerase, deionized water, react at 10-50°C for 0-24 hours; remove target DNA; precipitate RNA; centrifuge, discard supernatant, dissolve RNA precipitate, and obtain DIG-containing UTP-labeled antisense RNA strand; by Northern hybridization, the DIG-UTP-labeled antisense RNA strand is used as a probe to detect the target siRNA pool in transgenic plants. The invention has high specificity, simple operation and low cost, and can greatly reduce the detection cost of the target siRNA pool.

Description

A method for detecting target siRNA pool in transgenic plants

technical field

The invention relates to a method for detecting a target siRNA pool in transgenic plants.

Background technique

Gene interference is an important means of studying plant gene functions, and interference with normal ribonucleic acid accumulation (RNAi) is a method of gene interference. RNAi is a powerful experimental tool that uses homologous double-stranded RNA (dsRNA) to induce sequence-specific silencing of target genes, inhibiting or blocking gene expression. Small interfering RNA (siRNA) is 21-25 nucleotides (nt) formed by cutting dsRNA. It is an intermediate product in the RNAi pathway and a necessary factor for RNAi to exert its effect. Using transgenic plants to express dsRNA, the expressed dsRNA can form a target siRNA pool through the action of Dicer and Rde-1.

Detecting the formation of target siRNA pools in transgenic plants is of great significance for studying the interference process of target genes. At present, there are two main methods for detecting the target siRNA pool in transgenic plants: 1. using DNA probes containing radioactive phosphorus ³² P-dCTP; 2. detecting target siRNA using synthetic small antisense single-stranded RNA (ssRNA) ( Usually less than 30nt), and then use chemical modification method to add digoxigenin (DIG) tag at the 5' or 3' end of ssRNA. However, the implementation of these two methods has relatively large limitations: For method 1: the country has strict restrictions on the use of radioactive materials, and the use of radioactive probes requires complex experimental equipment and protective measures, and only a few units have the ability to operate radioactive materials. However, this method cannot be widely promoted and used, and it is expensive to entrust a qualified unit to conduct detection; for method 2: the method of chemical modification to add DIG tags is expensive, and there are a large number of target siRNAs in the target siRNA pool. Sense single-stranded RNA as a probe is prone to off-target effects. If a large amount of antisense single-stranded RNA is synthesized, the method of increasing DIG through chemical modification is expensive and difficult to implement.

Contents of the invention

The object of the present invention is to provide a method for detecting the target siRNA pool in transgenic plants, which uses DIG-labeled RNA probes to detect the target siRNA pool in transgenic plants. The method is highly specific, simple to operate, low in cost, and greatly reduces Assay cost for the pool of targeted siRNAs.

The present invention is realized based on the following theory and technology: the dsRNA of the target gene is randomly sheared into 21-25nt target siRNA in transgenic plants, and these target siRNAs with different nucleotide sequences form a target siRNA pool. In the present invention, the antisense RNA strand amplified by the target gene is complementary to the sense strand of each target siRNA in the target siRNA pool, and the antisense RNA strand can be combined with each target siRNA during the Northern hybridization process, as shown in Figure 1 , improved detection specificity and sensitivity.

In order to achieve the above object, the technical scheme of the present invention is as follows:

A method for detecting target siRNA pools in transgenic plants, comprising the steps of:

(1) Prepare the DIG-UTP-labeled antisense RNA strand using the target gene DNA as a template:

①The reaction system includes: UTP, ATP, GTP, CTP, DIG-UTP, RNA polymerase buffer, target DNA, RNA polymerase, deionized water, react at 10-50°C for 0-24 hours.

② Then, add DNase to the reaction system and react at 10-50°C to remove the target DNA in the reaction system.

③ Then, add deionized water and lithium chloride solution, and precipitate RNA at -20-0°C.

④ Centrifuge, discard the supernatant, add deionized water to dissolve the RNA precipitate, and obtain a high-purity target antisense RNA strand containing multiple DIG-UTP tags.

(2) Use the DIG-UTP-labeled antisense RNA strand as a probe to detect the target siRNA pool in transgenic plants:

①Perform the target gene RNA on polyacrylamide gel electrophoresis, then transfer the target gene RNA from the polyacrylamide gel to the nitrocellulose filter membrane, and dry the filter membrane.

② Then, after pre-hybridization of the dried filter membrane, add the DIG-UTP-labeled high-purity target antisense RNA strand obtained in step 1), and hybridize overnight.

③ Then, wash the filter membrane, develop color, expose, and image.

Preferably, a method for detecting target siRNA pools in transgenic plants, comprising the steps of:

(1) Prepare the DIG-UTP-labeled antisense RNA strand using the target gene DNA as a template:

①Reaction system: 50～200mM UTP, 50～200mM ATP, 50～200mM GTP, 50～200mM CTP, 5～20mM DIG-UTP, 10×RNA polymerase buffer, 1～20μl target DNA, 1～10μl RNA polymerase Enzyme, 0-20 μl deionized water, 25-45°C, react for 0-10 hours.

② Then, add DNase to the reaction system, react at 25-40°C for 0-1 hour, and remove the target DNA in the reaction system.

③ Add deionized water and lithium chloride solution, and precipitate RNA at -20-0°C.

④ Centrifuge at high speed for more than 5 minutes at 0-25°C, discard the supernatant, add deionized water to dissolve the RNA precipitate, and obtain high-purity target antisense RNA strands containing multiple DIG-UTP tags.

(2) Use the DIG-UTP-labeled antisense RNA strand as a probe to detect the target siRNA pool in transgenic plants:

① Conduct polyacrylamide gel electrophoresis on RNA, use a semi-dry electroporation system, transfer RNA from polyacrylamide gel to nitrocellulose filter membrane, and dry the filter membrane.

② Put the filter membrane into the hybridization solution, pre-hybridize at 55-65°C for more than 10 minutes, take out the filter membrane, immerse it in fresh hybridization solution, and add the DIG-UTP-labeled high-purity target antisense RNA probe obtained in step 1) , 37 ~ 45 ° C hybridization for more than 5 hours.

③ Slowly wash the filter membrane with low-stringency and high-stringency buffers in sequence, then soak the filter membrane in the blocking solution and antibody mixed solution, and use the washing solution to wash the filter membrane. Finally, soak the filter membrane with detection solution and chromogenic solution. After color development, put the filter membrane into the chromogenic imaging system, expose, take and save the imaging picture.

More preferably, a method for detecting target siRNA pools in transgenic plants, comprising the steps of:

(1) Prepare the DIG-UTP-labeled antisense RNA strand using the target gene DNA as a template:

① Reaction system: 1.7μl UTP (100mM), 1.7μl ATP (100mM), 1.7μl GTP (100mM), 1.7μl CTP (100mM), 1.0μl DIG-UTP (10mM), 2.0μl 10×RNA polymerase buffer , 5.0 μl target DNA, 2.0 μl RNA polymerase, 3.2 μl deionized water, 37°C, react for 4 hours.

② Then, add 1 μl DNase to the reaction system, and react at 37°C for 15 minutes to remove the target DNA in the reaction system.

③Add deionized water and lithium chloride solution, and precipitate at -20°C for 1 hour.

④ Centrifuge at 12,000 rpm for 15 minutes at 4°C. Discard the supernatant, add 50 μl deionized water to dissolve the RNA pellet, and obtain a highly pure target antisense RNA strand containing multiple DIG-UTP tags.

(2) Use the DIG-UTP-labeled antisense RNA strand as a probe to detect the target siRNA pool in transgenic plants:

① RNA transfer: perform polyacrylamide gel electrophoresis on RNA, use a semi-dry electroporation system, transfer RNA from polyacrylamide gel to nitrocellulose filter membrane, and dry the filter membrane.

② Put the filter membrane into the hybridization solution, pre-hybridize at 58°C for 30 minutes, take out the filter membrane, immerse it in fresh hybridization solution, add the DIG-UTP-labeled high-purity target antisense RNA probe obtained in step 1), and keep at 42°C Hybridize overnight.

③ Slowly wash the filter membrane with low-stringency and high-stringency buffers in sequence, then soak the filter membrane in the blocking solution and antibody mixed solution, and use the washing solution to wash the filter membrane. Finally, soak the filter membrane with detection solution and chromogenic solution. After color development, put the filter membrane into the chromogenic imaging system, expose, take and save the imaging picture.

The invention provides an RNA amplification kit comprising the DIG-UTP labeled antisense RNA chain.

Also, an application of an RNA amplification kit comprising the DIG-UTP-labeled antisense RNA strand in detecting a target siRNA pool in a transgenic plant.

In the present invention, the reaction system and reaction conditions selected in step (1) to prepare the DIG-UTP-labeled antisense RNA chain can ensure the generation of full-length target RNA containing DIG-label, and then effectively bind each siRNA in the siRNA pool, and Under these conditions, the efficiency of RNA production is the highest.

In step (2), the pre-hybridization at 55-65° C. can fully remove the RNA that is not bound to the filter membrane, improve the specificity of hybridization, and enable the siRNA to open double strands. Hybridize overnight at 37-45°C to allow the probe to fully contact and bind to the target siRNA, and once bound, it is not easy to separate.

Beneficial effects of the present invention:

1. The present invention utilizes DIG-labeled nucleic acid to obtain DIG-UTP-labeled antisense RNA strand and its RNA amplification kit, which is used to detect the target siRNA pool in transgenic plants. There is no radioactivity in the detection process, and it can be used in general biological laboratories. It can be synthesized, requires less equipment than making radioactive probes, does not require special protective measures, and has low cost.

2. Compared with the method of chemical modification, DIG-UTP-labeled antisense RNA strand and RNA amplification kit can be used multiple times, and this method of synthesizing antisense RNA strand greatly reduces the cost.

3. The invention has high specificity and simple operation.

Description of drawings

Fig. 1 is a schematic diagram of the principle of the detection method of the present invention.

Fig. 2 is an ethidium bromide staining diagram of analyzing the total RNA content in a sample using 1% agarose gel in Example 3 of the present invention.

Fig. 3 is a Northern Blot detection diagram corresponding to Fig. 2 .

Fig. 4 is an ethidium bromide staining diagram of analyzing the total content of small RNA in a sample using 15% polyacrylamide gel in Example 5 of the present invention.

Fig. 5 is a Northern Blot detection diagram using DIG-UTP antisense RNA probe in Example 5 of the present invention.

Fig. 6 is a Northern Blot detection diagram using a radioactive DNA probe in Example 6 of the present invention.

In Figures 2 to 6, lane 1 is RNA sample No. 1 (non-transgenic plant strain), and lanes 2-3 correspond to RNA sample No. 2 (transgenic plant strain) and RNA sample No. 3 (transgenic plant strain).

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

Unless otherwise specified, the following examples refer to the methods described in "Molecular Cloning Experiment Guide (Third Edition)" (Science Press, 2002). In the following examples, primer DNA synthesis and DNA sequencing were entrusted to Shanghai Sangon Bioengineering Co., Ltd. Chemical reagents (Trizol, various buffers, chloroform, etc.), molecular biology kits and enzymes were purchased from Hangzhou Nantian Biotechnology Co., Ltd. The glass, plastic products, and metal equipment used can be sterilized after soaking in 0.1% DEPC water overnight, and the glass and metal equipment can also be dry-baked at 180°C for 6 hours to remove contaminated RNase.

The preparation of embodiment 1 transgenic plant

In order to prove the versatility of the detection method in the present invention in terms of plants and genes, the model plant Arabidopsis is used as a transgenic plant to express dsRNA in the present embodiment, and the dsRNA gene is a model gene green fluorescent protein (GFP) gene, and the GFP sequence is as SEQ ID No:1 shown.

Agrobacterium transformation T-DNA carrier is constructed based on the pCambia 1300 vector (Cambia company), and the specific experimental steps are as follows:

1) Obtain DNA fragments that can be used to express GFP dsRNA

The nucleotide sequence of the GFP fragment was amplified from the pEGFP-N1 vector (Clontech Company), and restriction sites were designed at both ends of the primer:

GFPSF-BamH1: 5'GTGGGATCC atgagtaaaggagaagaact, the 5' end is designed with a BamHI site

GFPSR-EcoR1: 5'GTGGAATTC tttgtatagttcatccatgc, the 5' end is designed with an EcoRI site

GFPSF-Sac1: 5'GTGGAGCTC atgagtaaaggagaagaact, the 5' end is designed with a SacI site

GFPSR2-EcoR1: 5'GTGGAATTC gccattctttggtttgtctc, the 5' end is designed with an EcoRI site

Use GFPSF-BamH1 and GFPSR-EcoR1 as a pair of primers, use the pEGFP-N1 vector as a template to amplify, purify the PCR product to obtain a DNA fragment, and then carry out BamH1/EcoR1 digestion and purification to obtain a purified DNA fragment, named For GFP-sense.

Use GFPSF-Sac1 and GFPSR2-EcoR1 as a pair of primers, use the pEGFP-N1 vector as a template to amplify, purify the PCR product to obtain a DNA fragment, and then carry out Sac1/EcoR1 digestion and purification to obtain a purified DNA fragment, named For GFP-anti-sense.

The pUCmT vector (purchased from Shanghai Bioengineering Co., Ltd.) was digested with BamH1 and Sac1 and purified to obtain a DNA fragment, which was named TBS.

GFP-sense, GFP-anti-sense and TBS were ligated, and the ligated product was transformed into the cloning strain Escherichia coli TG1, and the plasmid T-dsGFP was obtained through identification. The plasmid T-dsGFP was digested with BamH1 and Sac1 to obtain the nucleotide fragment dsGFP.

2) Obtain promoter and terminator nucleotide fragments that can be used for construction.

The 35S promoter (its sequence is shown in SEQ ID NO: 2) and the terminator (its sequence is shown in SEQ ID NO: 3) are obtained from the pCambia 1300 vector, and the primers used are respectively:

35PF-Hind: 5'GCGAAGCTTgcacgacactctcgtctact, the 5' end is designed with a HindIII site

35PR-BamH: 5'GTGGGATCCagctcgagagagatagatttg, the 5' end is designed with a BamHI site

35TF-Sac1: 5'GTGGAGCTCtccttcgcaagacccttcct, the 5' end is designed with a SacI site;

35TR-Kpn1: 5'GTGGGTACCtggattttagtactggattt, the 5' end is designed with a KpnI site.

Using 35PF-Hind and 35PR-BamH as a pair of primers, the pCambia 1300 vector was used as a template for amplification, and the amplified PCR product was purified to obtain a DNA fragment, which was then digested with Hind3/BamH and purified to obtain the purified DNA fragment, named 35P.

Use 35TF-Sac1 and 35TR-Kpn1 as a pair of primers, use the pCambia 1300 vector as a template to amplify, purify the amplified PCR product to obtain a DNA fragment, and then perform Sac1/Kpn1 digestion and purification to obtain purified DNA Fragment, and named 35T.

3) Splice dsGFP and 35T fragments

The pUCmT vector was digested with BamH1 and Kpn1 and purified to obtain a DNA fragment, which was named TBK. The dsGFP, 35T and TBK were ligated, and the ligated product was transformed into the clone strain Escherichia coli TG1, and the plasmid T-dsGFP-T was obtained through identification. The plasmid T-dsGFP-T was digested with BamH1 and Kpn1 to obtain the nucleotide fragment dsGFP-T.

4) T-DNA vector construction

The pCambia 1300 vector was digested with HindIII and KpnI to obtain the nucleotide fragment 1300-HK, the 1300-HK, 35P and dsGFP-T were ligated, and the ligated product was transformed into the cloning strain Escherichia coli TG1. Bacillus plant transformed with plasmid 1300-35-dsGFP containing the T-DNA vector.

The plasmid 1300-35-dsGFP was transferred into Agrobacterium strain LBA4044, and the Agrobacterium was used for plant transformation. The transformation of Arabidopsis thaliana was entrusted to Wuhan Double Helix Biotechnology Co., Ltd. to obtain transgenic Arabidopsis seeds.

The extraction of total RNA in the transgenic plant of embodiment 2

Total RNA was extracted from transgenic Arabidopsis tissues using the Trizol method. The specific operation steps of this example are as follows:

1) Add transgenic Arabidopsis tissue and liquid nitrogen into a mortar and grind thoroughly.

2) Transfer 50 mg of the ground powder to a 2ml centrifuge tube, add 1mL Trizol to the centrifuge tube, vibrate vigorously for 30 seconds, place it on ice for 15 minutes, and centrifuge at 12000r/min at 4°C for 15 minutes.

3) Take the aqueous phase (located in the upper layer) obtained in step 2), add an equal volume of phenol/chloroform (1:1) to mix, shake vigorously for 10 minutes, and centrifuge at 12000rpm at 4°C for 15 minutes.

4) Take the aqueous phase (located in the upper layer) obtained in step 3), add an equal volume of chloroform, shake vigorously for 10 minutes, and then centrifuge as above (12000 rpm at 4° C. for 15 minutes).

5) Take the aqueous phase (located in the upper layer) obtained in step 4), and repeat step 3)+step 4).

6) Add an equal volume of pre-cooled isopropanol (or isoamyl alcohol) to the aqueous phase (located in the upper layer) obtained in step 5) and mix well, place at minus 20°C for 20 minutes, then centrifuge as above (4°C, 12000rpm for 15 minute).

7) The precipitate obtained in step 6) was washed twice with 70% ethanol, dried at room temperature for 10 minutes, dissolved in 50 ml of DEPC-treated deionized distilled water to dissolve RNA, aliquoted, and frozen at -20°C.

8) RNA quality and concentration detection. Take 2 μl of the RNA extracted in step 7), and detect the absorbance values at 260nm and 280nm, which are respectively 260nm value (OD260)=5.1, 280nm value (OD280)=2.6, OD260/OD280=1.96. According to the RNA purity standard, OD260/OD280 between 1.8-2.0 is high-purity RNA, so this embodiment obtained high-purity RNA. According to the calculation formula of RNA concentration (μg/mL): OD260 value×40ng/μl, the RNA concentration obtained in this example is 204ng/μl, which meets the standard of the follow-up experiment.

The detection of dsRNA in the transgenic plant of embodiment 3

In order to confirm that the dsRNA has been expressed in the plant, the production of the dsRNA in the plant was detected by Northern hybridization.

Use the method of Example 2 to extract the total RNA of the leaves of 1 non-transgenic crop and 2 different strains of transgenic plants, and the numbers are respectively No. 1 RNA (non-transgenic), No. 2 RNA (transgenic), and No. 3 RNA (transgenic) .

The specific operation steps are as follows:

1) In a centrifuge tube, mix 10 μl RNA sample No. 1, RNA sample No. 2, and RNA sample No. 3 with 5 μl RNA loading buffer respectively to obtain RNA loading solutions No. 1, No. 2, and No. 3.

2) Cover the centrifuge tube tightly, incubate the RNA sample solution at 90°C for 5 minutes, and immediately place the sample on ice for 10 minutes to convert dsRNA into single-stranded RNA. Centrifuge the RNA loading solution at 10,000 rpm for 5 seconds to allow all the liquid in the tube to settle to the bottom of the tube.

3) Pouring a 1% agarose horizontal gel to analyze the total RNA content in the RNA loading solution. Add 50ml of 1×TBE as a buffer and 5g of agarose in a conical flask, heat it in a microwave oven, after the agarose is dissolved, add 50μl of 0.5mg/ml ethidium bromide solution, and pour the agarose solution into a gel plate , cooled and solidified to form a 1% agarose gel. The electrophoresis tank used for RNA electrophoresis needs to be washed with detergent solution, rinsed with water, dried with ethanol, then filled with 3% H ₂ O ₂ , and left at room temperature for 10 minutes, then rinse the electrophoresis tank thoroughly with DEPC-treated water . Add RNA samples No. 1-3 to the gel 1-3 sample wells, immerse the gel in 1×TBE electrophoresis solution, and perform electrophoresis at a voltage of 3-4V/cm. After the electrophoresis, the photos of RNA staining were taken under ultraviolet light (as shown in Figure 2). In Fig. 2, the RNA bands in each lane are clear and distinct, without a large number of fuzzy dragging bands. This shows that the RNA is not degraded in the RNA loading solution, and the brightness of the sample RNA bands in the three swimming lanes is consistent, indicating that the loading amount of RNA in the samples in the three swimming lanes is consistent.

4) Move the gel after electrophoresis in step 3) to a glass dish, use a sharp blade to repair the useless part of the gel, and cut off a corner at the upper left corner of the gel (the end of the sample hole is the upper side), as the following operation Marking of gel orientation during the process.

5) Pour 20ml of depurination buffer (0.2M HCl) into a glass dish and shake at room temperature for 20 minutes.

6) Remove the depurination buffer in the glass dish, pour 20ml of denaturation buffer (50mM NaOH, 1.5M NaCl), and shake at room temperature for 20 minutes.

7) Remove the denaturation buffer in the glass dish, pour 20ml neutralization buffer (1M Tris, 1.5M NaCl, pH 7.4), and shake at room temperature for 20 minutes.

8) Use plexiglass as a platform, place it on a plastic square box, and put a piece of Whatman 3MM filter paper on the plexiglass. Pour 20×SSC (3M NaCl, 0.3M sodium citrate,pH 7.0) into the plastic square box so that the liquid level is slightly lower than the surface of the platform. When the 3MM filter paper above the platform is wetted by capillary action, drive it out with a glass rod All bubbles.

9) Float the nitrocellulose filter membrane on the surface of deionized water until the filter membrane is soaked from bottom to top, then move the filter membrane to 20×SSC and soak for 5 minutes, cut off a corner of the filter membrane with a clean scalpel blade, Make it correspond to the cut corner of the gel.

10) Place the gel in the center of Whatman 3MM filter paper, and use a glass rod to drive out all air bubbles. Place a warm nitrocellulose filter over the gel so that the cut corners of the two overlap. One edge of the filter should just extend beyond the edge of the top line of the injection well on the gel.

11) Soak two pieces of Whatman 3MM filter paper of the same size as the gel with 2×SSC solution (dilute 20×SSC 10 times with deionized water), and place it warmly on top of the wet nitrocellulose filter. Use a glass rod to drive out air bubbles. Place a stack (5-8cm high) of paper towels slightly smaller than 3MM filter paper on top of Whatman 3MM filter paper, place a glass plate on top of the paper towels, and press the glass plate with a weight of 500g. In this way, RNA was transferred from the gel to the filter by capillary action, and the transfer continued for 18 hours.

12) After step 11), remove the paper towel and Whatman 3MM filter paper above the nitrocellulose filter, take out the nitrocellulose filter, soak the filter in 2×SSC solution, take out the filter after 5 minutes and place it flat On a piece of Whatman 3MM filter paper, dry bake at 120°C for 30 minutes.

13) Preparation of GFP probe containing DIG label

Primers were designed according to the interference gene GFP for PCR amplification. The primer sequences are as follows:

GFP-F:ATGAGTAAAGGAGAAGAACT

GFP-R: TTTGTATAGTTCATCCATGC

The PCR reaction system is: 50 μl 2х high-fidelity polymerase (PFU) Mix, 4 μl GFP-F (10 μM), 4 μl GFP-R (10 μM), 1 μl GFP DNA, 41 μl deionized water (ddH ₂ O), the total volume is 100 μl; The PCR reaction program is: 1.95°C, 10 minutes; 2.95°C, 40 seconds; 3.52°C, 40 seconds; 4.68°C, 2 minutes; 5. Steps 2-4, 25 cycles; 6.68°C, 10 minutes; 7.16°C, save . The DNA sequence of GFP was amplified by PCR, and the PCR product purification kit was used to purify and obtain the DNA of GFP, and the DNA was diluted to 100 ng/μl. Using this DNA as a template, 100 μl of DIG-labeled GFP probe was prepared using the Roche DIG-labeled Probe Synthesis Kit.

14) Immerse the filter membrane in step 12) into 10ml DIG Easy Hyb solution (Roche NorthernBlot kit), and pre-hybridize at 42°C for 30 minutes. Take out the filter membrane and immerse it in fresh 10ml DIG Easy Hyb solution, add 10μl of GFP probe prepared in step 13), and hybridize overnight at 42°C.

15) At room temperature, soak the filter membrane hybridized overnight in 20 ml of low stringency buffer (2×SSC, 0.1% SDS) twice, 15 minutes each time. The filter membrane was taken out, and soaked twice in 20 ml of high stringency buffer (0.1×SSC, 0.1% SDS) at 65° C. for 15 minutes each time. The filter membrane was taken out, and soaked twice in 20ml washing solution (0.1M maleic acid, 0.15M NaCl, pH 7.5, 0.3% Tween) at room temperature for 5 minutes each time. The filter membrane was taken out, and incubated in 15 mL blocking solution (Roche Northern Blot kit) for 30 minutes at room temperature. The filter membrane was taken out, and immersed in a mixed solution of 5 ml of blocking solution and 10 μl of DIG antibody (Roche Northern Blot kit) for 30 minutes at room temperature. The filter membrane was taken out, and soaked twice in 20ml washing solution (0.1M maleic acid, 0.15M NaCl, pH 7.5, 0.3% Tween) at room temperature for 15 minutes each time. Take out the filter membrane and soak it in 15mL detection solution (0.1M Tris-HCl, 0.1M NaCl, pH 9.5) for 5 minutes at room temperature. Take out the filter membrane and soak it in 1ml CSPD ready-to-use (Roche Northern Blot Kit) at 37°C for 10 minutes.

16) Put the filter membrane in step 15) into a chemiluminescence chromogenic imaging system (model: Tanon-6600), expose for 2 minutes, take and save the imaging picture (as shown in Figure 3).

In this embodiment, the total RNA of the sample is bound to the filter membrane. If the sample contains dsRNA of GFP, the GFP DNA probe with DIG label will specifically bind to the dsRNA of GFP and be immobilized on the filter membrane. A black band appears in the imaging system. If the sample does not have GFP dsRNA, the DIG-labeled GFP DNA probe cannot bind to non-specific RNA, and cannot be immobilized on the filter membrane, and finally no bands will be displayed in the chemiluminescent chromogenic imaging system.

As can be seen from Figure 3, No. 1 RNA sample has no black band, indicating that there is no GFP dsRNA in No. 1 RNA sample, which is consistent with the fact that No. 1 RNA sample is non-transgenic Arabidopsis; No. 2 and No. 3 RNA samples are both There are black bands, indicating that RNA samples No. 2 and No. 3 contain dsRNA of GFP, indicating that transgenic Arabidopsis No. 2 and No. 3 express dsRNA of GFP.

The expression level of dsRNA in plants is related to the insertion site of T-DNA in the plant genome. In Figure 3, the black band of No. 3 sample is obviously larger than that of No. 2 sample, indicating that the dsRNA of GFP in No. 3 sample The content is greater than the content of dsRNA of GFP in No. 2 sample.

Embodiment 4 prepares the antisense RNA chain comprising DIG-UTP

With the 100ng/μl GFP DNA obtained in step 13) in Example 3 as a template, prepare the antisense GFP RNA chain comprising DIG-UTP, and the specific steps are as follows:

1) Use the following reaction system: 1.7μl UTP (100mM), 1.7μl ATP (100mM), 1.7μl GTP (100mM), 1.7μl CTP (100mM), 1.0μl DIG-UTP (10mM), 2.0μl 10×RNA polymerization Enzyme buffer (100mM NaCl, 5mM DTT, pH7.4), 5.0μl GFP DNA, 2.0μl RNA polymerase, 3.2μl deionized water, 37°C, react for 4 hours. In this reaction system, DIG-UTP will partially replace conventional UTP to obtain GFP antisense RNA containing multiple DIG-UTP tags.

2) Add 1 μl DNase to the system in step 1), react at 37°C for 15 minutes, and remove the DNA in the reaction system. Add 15 μl deionized water and 15 μl lithium chloride solution, and precipitate at -20°C for 1 hour.

3) Centrifuge at 12000 rpm for 15 minutes at 4°C. Discard the supernatant and add 50 μl deionized water to dissolve the RNA pellet.

4) RNA quality and concentration detection: get 2 μl of the RNA extracted in step 3), and detect its absorbance at 260nm and 280nm, which are respectively: 260nm value (OD260)=2.6, 280nm value (OD280)=1.4, OD260/OD280= 1.85. According to the standard of RNA purity, OD260/OD280 is high-purity RNA between 1.8-2.0, so high-purity RNA was obtained in this embodiment. According to the calculation formula of RNA concentration (μg/mL): OD260 value×40ng/μl, the RNA concentration obtained in this example is 104ng/μl, which meets the standard of subsequent experiments.

In this example, a high-purity GFP antisense RNA containing multiple DIG-UTP markers was obtained, which was named GFP-DIG-antiRNA.

Example 5 Using GFP-DIG-antiRNA as a probe to detect the target siRNA pool in transgenic plants

Use 30 μl of transgenic plant total RNA samples obtained in Example 3: RNA No. 1, RNA No. 2, and RNA No. 3 to detect the content of the target siRNA pool. The specific operation steps are as follows:

1) Prepare 15% polyacrylamide gel with 1×TBE as buffer. The electrophoresis tank used for RNA electrophoresis needs to be washed with detergent solution, rinsed with water, dried with ethanol, then filled with 3% (volume %) H ₂ O ₂ , left at room temperature for 10 minutes, then washed with DEPC-treated water Rinse the electrophoresis tank thoroughly. Add No. 1, No. 2 and No. 3 total RNA samples to the sample wells of 15% polyacrylamide gel gel respectively, and immerse the gel in 1×TBE electrophoresis solution (0.09mol/L Tris-boric acid, 0.002mol/L EDTA ), electrophoresis was performed with a current of 30-45mA.

2) After electrophoresis, soak the polyacrylamide gel in a solution containing 0.5ug/ml ethidium bromide for 5 minutes. Photos of RNA staining were taken under ultraviolet light (as shown in Figure 4). In Figure 4, the RNA bands of No. 1, No. 2 and No. 3 are clear, the outline is obvious, and there is no large amount of fuzzy dragging bands, indicating that the RNA has not been degraded in the RNA sample solution, and the brightness of the RNA bands in the three samples is consistent, indicating that The loading amount of RNA in the 3 samples was the same.

3) Using a semi-dry electroporation system (model: BioRad Trans-Blot), transfer the RNA from the polyacrylamide gel to the nitrocellulose filter. During electroporation, use 0.25×TBE buffer solution (1×TBE electrophoresis solution diluted 4 times), 100mA current for 1 hour.

4) Take out the nitrocellulose filter membrane, soak the filter membrane in 2×SSC solution for 5 minutes, take out the filter membrane and place it flat on a piece of Whatman 3MM filter paper, dry bake at 120°C for 30 minutes.

5) Immerse the filter membrane in step 4) in 10ml DIG Easy Hyb solution (Roche NorthernBlot kit), and pre-hybridize at 58°C for 30 minutes.

6) Take out the filter membrane and immerse it in 10 ml of fresh DIG Easy Hyb solution, add 10 μl of the GFP-DIG-antiRNA probe prepared in Example 3, and hybridize overnight at 42°C.

7) At room temperature, soak the filter membrane in step 6) in 20 ml of low stringency buffer (2×SSC, 0.1% SDS) for 2 times, 15 minutes each time. The filter membrane was taken out, and soaked twice in 20 ml of high stringency buffer (0.1×SSC, 0.1% SDS) at 65° C. for 15 minutes each time. The filter membrane was taken out, and soaked twice in 20ml washing solution (0.1M maleic acid, 0.15M NaCl, pH 7.5, 0.3% Tween) at room temperature for 5 minutes each time. The filter membrane was taken out, and incubated in 15 mL blocking solution (Roche Northern Blot kit) for 30 minutes at room temperature. The filter membrane was taken out, and immersed in a mixed solution of 5 ml of blocking solution and 10 μl of DIG antibody (Roche Northern Blot kit) for 30 minutes at room temperature. The filter membrane was taken out, and soaked twice in 20ml washing solution (0.1M maleic acid, 0.15M NaCl, pH 7.5, 0.3% Tween) at room temperature for 15 minutes each time. Take out the filter membrane and soak it in 15mL detection solution (0.1M Tris-HCl, 0.1M NaCl, pH 9.5) for 5 minutes at room temperature. Take out the filter membrane and soak it in 1ml CSPD ready-to-use (Roche Northern Blot Kit) at 37°C for 10 minutes.

8) Put the filter membrane in step 7) into a chemiluminescence chromogenic imaging system (model: Tanon-6600), expose for 2 minutes, take and save the imaging picture (as shown in Figure 5).

In this embodiment, the total RNA of the sample is bound to the filter membrane. If the sample contains the siRNA pool of GFP, the antisense RNA probe of the DIG-UTP of GFP will specifically bind to the siRNA pool of GFP and be immobilized on the filter membrane (as shown in FIG. 1), and finally showed black bands in the chemiluminescent chromogenic imaging system. If the sample does not have a GFP siRNA pool, the GFP DIG-UTP antisense RNA probe cannot bind to the non-specific siRNA pool, and cannot be immobilized on the filter membrane, and finally no bands are displayed in the chemiluminescent chromogenic imaging system.

As can be seen from Figure 5, No. 1 RNA sample has no black band, indicating that there is no GFP siRNA pool in No. 1 RNA sample, which is consistent with the fact that No. 1 RNA sample is non-transgenic Arabidopsis; No. 2 and No. 3 RNA samples are both There are black bands, indicating that No. 2 and No. 3 samples contain the RNA pool of GFP, and the result shows that No. 2 and No. 3 transgenic Arabidopsis have produced the siRNA pool of GFP. Therefore, the probe designed by the present invention can specifically detect to these siRNA pools.

Example 6 Verification using radioactive materials ³² P

In this experiment, radioactive material ³² P was used to verify the accuracy of the results in Example 5. The specific operation steps of this example are as follows:

1)-5): Same as steps 1)-5) in Example 5.

6) Preparation of ³² P probe for GFP: 100 ng/μl GFP DNA prepared in step 13) of Example 3 was used as a template, denatured at 95° C. for 5 minutes, and kept in an ice bath for 5 minutes. The reaction system was as follows (using Promega, Prime-a-Gene Labeling kit): 0.5 μl dATP (100 mM), 0.5 μl dTTP (100 mM), 0.5 μl dGTP (100 mM), 0.5 μl dCTP (100 mM), 2.0 μl BSA (10 mg/ml ), 10 μl 5×buffer, 1.0 μl Klenow enzyme (5u/μl), 5.0 μl ³² P-dCTP, 5.0 μl GFP DNA (100ng/μl), 25 μl deionized water; react at room temperature for 1 hour, denature at 95°C for 5 minutes , ice-bathed for 5 minutes to obtain the GFP- ³² P probe.

7) Take out the filter membrane and immerse it in fresh 10ml DIG Easy Hyb solution, add 10μl of GFP ^-32P probe, and hybridize overnight at 42°C.

8) At room temperature, soak the filter membrane in step 7) in 20 ml of low stringency buffer (2×SSC, 0.1% SDS) for 2 times, 15 minutes each time. The filter membrane was taken out, and soaked twice in 20 ml of high stringency buffer (0.1×SSC, 0.1% SDS) at 55° C. for 15 minutes each time.

9) Rinse the filter membrane with deionized water for 1 minute, and absorb the water on the membrane with filter paper. Wrap the film with thin plastic paper, put it in a dark box, and press the X-ray film in the dark room. Cassette autoradiography was performed for 3 days, and the imaging pictures were saved (as shown in FIG. 6 ).

The bands in Figure 6 appear due to the ³² P luminescence in the probe. In this embodiment, the total RNA of the sample is bound to the filter membrane. If the sample contains the GFP siRNA pool, the GFP ^-32P probe will specifically bind to the GFP siRNA pool and immobilize on the filter membrane, finally showing black bands in the dark room. If there is no GFP siRNA pool in the sample, the GFP ^-32P probe cannot be bound to the non-specific siRNA pool and cannot be immobilized on the filter membrane, and finally no bands are displayed in the dark room.

It can be seen from Figure 6 that RNA sample No. 1 has no black bands, indicating that there is no GFP siRNA pool in RNA sample No. 1; RNA samples No. 2 and No. 3 have black bands, indicating that samples No. 2 and No. 3 contain GFP siRNA pool of GFP, this result shows that No. 2 and No. 3 transgenic Arabidopsis thaliana produced the siRNA pool of GFP, which is the same as that in Example 5. It is verified that the method of the present invention using DIG-labeled RNA probes to detect target siRNA pools is feasible and accurate.

Claims (5)

1. A method for detecting target siRNA pool in transgenic plants, characterized in that, comprising the steps: 1) Prepare the DIG-UTP-labeled antisense RNA strand using the target gene DNA as a template: ①The reaction system includes: UTP, ATP, GTP, CTP, DIG-UTP, RNA polymerase buffer, target DNA, RNA polymerase, deionized water, react at 10-50°C for 0-24 hours; ② Then, add DNase to the reaction system, react at 10-50°C to remove the target DNA in the reaction system; ③ Then, add deionized water and lithium chloride solution, and precipitate RNA at -20-0°C; ④ Centrifuge, discard the supernatant, add deionized water to dissolve the RNA precipitate, and obtain a high-purity target antisense RNA strand containing multiple DIG-UTP tags; 2) Use the DIG-UTP-labeled antisense RNA strand as a probe to detect the target siRNA pool in transgenic plants: ①Perform the target gene RNA on polyacrylamide gel electrophoresis, then transfer the target gene RNA from the polyacrylamide gel to the nitrocellulose filter membrane, and dry the filter membrane; ② Then, after pre-hybridizing the dried filter membrane, add the DIG-UTP-labeled high-purity target antisense RNA strand obtained in step 1), and hybridize overnight; ③ Then, wash the filter membrane, develop color, expose, and image. 2. the method for detecting target siRNA pool in transgenic plants as claimed in claim 1, is characterized in that, comprises the steps: 1) Prepare the DIG-UTP-labeled antisense RNA strand using the target gene DNA as a template: ①Reaction system: 50～200mM UTP, 50～200mM ATP, 50～200mM GTP, 50～200mM CTP, 5～20mM DIG-UTP, 10×RNA polymerase buffer, 1～20μl target DNA, 1～10μl RNA polymerase Enzyme, 0~20μl deionized water, 25~45℃, react for 0~10 hours; ② Then, add DNase to the reaction system, react at 25-40°C for 0-1 hour, and remove the target DNA in the reaction system; ③Add deionized water and lithium chloride solution, and precipitate RNA at -20～0℃; ④ Centrifuge at 0-25°C for more than 5 minutes, discard the supernatant, add deionized water to dissolve the RNA precipitate, and obtain a high-purity target antisense RNA strand containing multiple DIG-UTP tags; 2) Use the DIG-UTP-labeled antisense RNA strand as a probe to detect the target siRNA pool in transgenic plants: ①Perform RNA on polyacrylamide gel electrophoresis, use a semi-dry electroporation system, transfer RNA from polyacrylamide gel to nitrocellulose filter membrane, and dry the filter membrane; ②Then, put the filter membrane into the hybridization solution, pre-hybridize at 55-65°C for more than 10 minutes, take out the filter membrane, immerse it in fresh hybridization solution, and add the DIG-UTP-labeled high-purity target antisense RNA obtained in step 1) Probes, hybridization at 37-45°C for more than 5 hours; ③ Then, use low stringency and high stringency buffers to slowly wash the filter membrane in sequence, then soak the filter membrane in the blocking solution and antibody mixed solution, and use the washing solution to wash the filter membrane; finally use the detection solution and color development Soak the filter membrane in the liquid, after color development, put the filter membrane into the color imaging system, expose, take and save the imaging picture. 3. the method for detecting target siRNA pool in transgenic plants as claimed in claim 1 or 2, is characterized in that, comprises the steps: 1) Prepare the DIG-UTP-labeled antisense RNA strand using the target gene DNA as a template: ①Reaction system: 1.7μl 100mM UTP, 1.7μl 100mM ATP, 1.7μl 100mM GTP, 1.7μl 100mM CTP, 1.0μl 10mM DIG-UTP, 2.0μl 10×RNA polymerase buffer, 5.0μl target DNA, 2.0μl RNA polymerase, 3.2 μl deionized water, 37°C, react for 4 hours; ② Then, add DNase to the reaction system and react at 37°C for 15 minutes to remove the target DNA in the reaction system; ③ Then, add deionized water and lithium chloride solution, and precipitate at -20°C for 1 hour; ④ Centrifuge at 12,000 rpm for 15 minutes at 4°C, discard the supernatant, add deionized water to dissolve the RNA precipitate, and obtain high-purity target antisense RNA strands containing multiple DIG-UTP tags. 2) Use the DIG-UTP-labeled antisense RNA strand as a probe to detect the target siRNA pool in transgenic plants: ①Perform RNA on polyacrylamide gel electrophoresis, use a semi-dry electroporation system, transfer RNA from polyacrylamide gel to nitrocellulose filter membrane, and dry the filter membrane; ② Then, put the filter membrane into the hybridization solution, pre-hybridize at 58°C for 30 minutes, take out the filter membrane, immerse in the fresh hybridization solution, add the DIG-UTP-labeled high-purity target antisense RNA probe obtained in step 1), Hybridize overnight at 42°C; ③ Then, use low stringency and high stringency buffers to slowly wash the filter membrane in turn, then soak the filter membrane in the blocking solution and antibody mixed solution, and use the washing solution to wash the filter membrane; finally use the detection solution and display Soak the filter membrane in the color solution, and after developing the color, put the filter membrane into the color imaging system, expose, take and save the imaging picture. 4. An RNA amplification kit comprising the DIG-UTP-labeled antisense RNA strand according to any one of claims 1-3. 5. The application of the RNA amplification kit as claimed in claim 4 in detecting the target siRNA pool in transgenic plants.