CN110699315A - Method for extracting fixed tissue protoplast nucleic acid - Google Patents
Method for extracting fixed tissue protoplast nucleic acid Download PDFInfo
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Abstract
The invention relates to a method for extracting protoplast nucleic acid of a fixed tissue. The method comprises the following steps: 1) obtaining young root tip tissues of corn; 2) tissue fixation; 3) preparing fixed corn root tip tissue protoplasts; 4) and (4) extracting nucleic acid. The material of the invention is easy to obtain, the process operation is simple, the number of the protoplasts obtained by separation is large, and the integrity of the extracted nucleic acid is good.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a nucleic acid extraction method, in particular to a method for fixing corn root tip tissues by using a fixing solution and carrying out enzymolysis on the corn root tip tissues into protoplasts, and respectively extracting DNA and RNA of the protoplasts by adopting CTAB (cetyl trimethyl ammonium bromide) and TRNzol (TRNzol) methods so as to establish a technical system for extracting the nucleic acid of the fixed tissue protoplasts.
Background
The preparation and separation and enrichment of protoplasts often take several hours, and when plant tissues are immersed in a protoplast separation solution after leaving the parent, the physiological activities and the transcription information in living cells change along with the change of environmental conditions, and even if the sequencing is successfully carried out, the transcription information on the target time point of a sample cannot be completely represented. Therefore, it is necessary to fix the tissue before cell isolation to ensure temporal characterization of the transcriptional information.
The fixation is a treatment process for keeping substance components or fine structures contained in a biological sample in an original state, and through the fixation, enzymes in tissues are quickly inactivated, so that autolysis in an isolated state is avoided, and the original structure and form are kept. Although there are many reports on the solutions and methods that can be used for tissue fixation, the materials fixed by these methods are mainly used for studies such as structural observation, and rarely for the purpose of extracting nucleic acids. Different fixation methods have no study on how to influence the protoplast separation and nucleic acid extraction of the fixed tissue.
Disclosure of Invention
The invention provides an accurate, reliable and basic fixed tissue protoplast nucleic acid extraction method for the research of subsequent transcription information aiming at the technical problems in the background technology.
The technical solution of the invention is as follows: the invention relates to a method for extracting protoplast nucleic acid of a fixed tissue, which is characterized by comprising the following steps: the method comprises the following steps:
1) obtaining young root tip tissues of corn;
selecting full and healthy corn seeds, soaking the corn seeds for 4-6 hours in ddH2O, after the seeds are fully swelled, intensively placing the seeds on a tray paved with 4 layers of wet gauze, covering a towel on the seeds to keep moisture, placing the seeds in a constant-temperature incubator at 28-36 ℃ for culturing for 4-5 days, and cutting young root tips with the front end of about 3cm for fixation;
2) tissue fixation;
2.1) weighing 1.8-2.2g of collected root tips, longitudinally cutting the root tips firstly, then transversely cutting the root tips into small sections with the length of 0.8-1.2cm, soaking the small sections in a fixing solution, and fixing the small sections at room temperature for 0.8-1.2 h;
2.2) pouring out the fixing solution, adding sterile water (extracting RNA, and then using DEPC treated water) to soak and wash for 3 times, 5min each time;
3) preparing fixed corn root tip tissue protoplasts;
3.1) putting the cleaned fixed tissue into the enzymolysis liquid, putting the fixed tissue into a shaking table, setting the temperature at 30-40 ℃, setting the rotating speed at 50-60r/min, carrying out enzymolysis for 4-6h, and carrying out dark treatment;
3.2) after the enzymolysis is finished, sucking the enzymolysis mixed solution by a Pasteur straw, filtering by a nylon mesh screen with the thickness of 0.45 mu m, taking the filtrate, centrifuging for 8-12min in a centrifugal tube at the speed of 1000-; adding 3-4 ml of cell suspension, centrifuging for 8-12min at 1000-; repeating the above step 2 times, adding 1ml of cell suspension to the final pellet;
3.3) purifying the protoplast; gently spreading the protoplast after enzymolysis in 75-90% sucrose solution, and centrifuging at 1200-1400r/min for 15-25min for purification;
4) nucleic acid extraction
4.1) DNA extraction:
4.1.1 after purification, the protoplast 2500-; keeping the temperature at 60-70 ℃ for more than 30min, and slightly shaking for 3-5 times every 10min in the heat preservation process;
4.1.2) taking out the centrifuge tube, cooling to room temperature, adding equal volume of phenol: chloroform: isoamyl alcohol (volume ratio is 25:24:1), namely 700-;
4.1.3) transfer 600. mu.l of supernatant into a new centrifuge tube, add equal volume of phenol: chloroform: isoamyl alcohol (volume ratio 25:24:1) 600 mul, shaking gently for 3-5min, centrifuging at 12000-14000rpm/min at 4 ℃ for 8-12 min;
4.1.4) taking 400 mul of supernatant again to a new centrifuge tube, adding 0.6 times volume of pre-cooled isopropanol, and standing for more than 30min at room temperature; 12000-;
4.1.5) opening the cover and standing, completely drying the residual alcohol at room temperature, adding 30 mu lddH2O, dissolving DNA in water bath at 65 ℃ for 5-10min, and preserving at-20 ℃;
4.2) RNA extraction:
4.2.1) centrifuging the purified protoplast at 3500rpm/min for 10min, removing the supernatant, adding 800-;
4.2.2) taking 850 mul of supernatant fluid 750-; 12000-; 12000 plus 14000rpm/min,4 ℃, centrifugation for 10min, supernatant removal, 1000 mul of 75 percent ethanol (RNase-Free ddH2O) washing precipitation 12000 plus 14000rpm/min,4 ℃, centrifugation for 3min, liquid pouring out, sucking out the residual small amount of liquid by a gun head after short centrifugation, standing for 3min at room temperature, adding RNase-Free dH2O 30 mul, repeatedly blowing and evenly mixing.
Preferably, the fixing solution in step 2.1) is Carnoy 2, wherein the ratio of ethanol: glacial acetic acid: chloroform 6:1:3, Methacarn, wherein methanol: glacial acetic acid: chloroform 6:1: 3; and (5) fixing the corn root tip tissue.
Preferably, the enzymolysis liquid in the step 3.1) is prepared into KH2PO4:27.2mg/L, KNO3:101.0mg/L, CaCl 2.2H2O: 1480mg/L, MgSO 4.7H 2O:246mg/L, CuSO 4.5H 2O:0.025mg/L, KI: 0.16mg/L, mannitol: 14%, cellulase: 2.8%, eductase: 0.6 percent and the balance of hydrosolvent, the PH value is adjusted to 5.8 after constant volume, and the filtration and sterilization are carried out at 0.45 mu m, and the preparation is prepared at present.
Preferably, the cell suspension in step 3.2) is formulated as KH2PO4:27.2mg/L, KNO3:101.0mg/L, CaCl 2.2H2O: 1480mg/L, MgSO 4.7H2O: 246mg/L, CuSO 4.5H 2O:0.025mg/L, KI: 0.16mg/L, mannitol: 14 percent and the balance of water solvent, and is filtered and sterilized by 0.45 mu m, and the preparation is prepared just before use.
Preferably, step 4) further comprises step 5) nucleic acid quality detection;
5.1) nucleic acid concentration detection: with NanoDropTMRespectively measuring DNA and RNA yields, 260nm and 280nm ultraviolet absorption values OD260 and OD280 and OD260/OD280 of samples obtained by three times of repetition by using a 2000 type spectrophotometer;
5.2) nucleic acid integrity testing;
the extracted nucleic acid products were subjected to gel electrophoresis with 1% agarose and image analysis by a gel imager of the GelDoc XR System type.
Preferably, the specific steps of step 5.2) are as follows:
DNA amplification: according to known maize gene ASPAT1.2, TUB, sh and zein sequences published in NCBI library, primers 1, 2, 3 and 4 with target fragment lengths of about 200 bp, 400 bp, 600 bp and 800bp are respectively designed by using online software Primer3(http:// Primer3.ut. ee /), and are synthesized by Shanghai biological engineering Co., Ltd; selecting the four genes to carry out PCR detection by taking the extracted DNA as a template; reaction system 25 u L, DNA 100ng,2 x Premix Taq12.5 u L, primer each 0.15mmol.L-1, ddH2O6.5 u L; PCR conditions of 95 ℃, 5min, 95 ℃,40 s, 50-58 ℃,40 s,72 ℃,50 s and 40 cycles; extending for 5min at 72 ℃; storing at 4 ℃, performing gel electrophoresis on the PCR amplification product by using 1% agarose, and analyzing images by using a GelDoc XR System type gel imager;
RNA amplification: according to the mRNA sequences of known maize genes ASPAT1.2, TUB and as published in NCBI library, primers 1, 5, 6 and 7 are designed by using online software Primer3(http:// Primer3.ut. ee /), and synthesized by Shanghai Bioengineering, Inc.; cDNA 1 st chain synthesis was performed according to the (FastQuant RT Kit (with gDNase)) instructions (TIANGEN); carrying out PCR detection on genes with different lengths by taking the reverse transcribed cDNA as a template; the reaction system is 25 mu L, cDNA3000ng,2 XPremix Taq12.5 mu L, the upstream and downstream primers are 0.15mmol L-1 and ddH2O 6.5.5 mu L respectively; the reaction procedure comprises the steps of 95 ℃, 5min, 95 ℃,40 s, 50-57.5 ℃,40 s,72 ℃,50 s and 40 cycles; 72 ℃ for 5 min; the PCR amplification product was stored at 4 ℃ and subjected to gel electrophoresis using 1% agarose, and image processing using a GelDoc XR System type gel imaging split System.
The method for extracting the fixed tissue protoplast nucleic acid takes the young tender tissue of the corn root tip as a material, and is completed by five steps of acquisition of the young root tip tissue of the corn, tissue fixation, preparation of the fixed protoplast of the corn root tip tissue, nucleic acid extraction and nucleic acid quality detection; the method has the advantages of easily-accessible material, simple operation, high protoplast amount, and good integrity of extracted nucleic acid.
Drawings
FIG. 1 protoplasts before purification;
FIG. 2 purified protoplasts;
FIG. 3DNA electrophoresis detection map;
FIG. 4 is an electrophoretic RNA detection scheme;
FIG. 5 is an amplification product of DNA;
FIG. 6 amplification product of RNA.
Detailed Description
The invention is further described below with reference to the figures and the specific examples.
Table 1 shows primers designed and synthesized according to the present invention and their base sequences:
maize reference genes for isolated nucleic acid integrity testing: zmaspat 1.2: an aspartate aminotransferase; ZmTUB: beta-tubulin; zmsh: a sucrose synthase; zmzein: a prolamin; ZmAS: ASR transcription factors (the same gene amplifies different segments in order to ensure the integrity of the gene and to compromise the efficiency of amplification.
The method of the first embodiment of the invention is as follows:
1) obtaining young root tip tissues of corn;
selecting full and healthy corn seeds, soaking the corn seeds for 5 hours in ddH2O, after the seeds are fully swelled, intensively placing the seeds on a tray paved with 4 layers of wet gauze, covering a towel on the seeds to keep moisture, placing the seeds in a constant-temperature incubator at 32 ℃ for culturing for 5 days, and cutting young root tips of about 3cm at the front end for fixation;
2) tissue fixation;
2.1) weighing 2g of the collected root tips, longitudinally cutting the root tips firstly, then transversely cutting the root tips into small sections with the length of about 1cm, and soaking the small sections in a fixing solution Carnoy 2 (ethanol: glacial acetic acid: chloroform 6:1:3), Methacarn (methanol: glacial acetic acid: chloroform 6:1:3), fixing for 1h at room temperature,
2.2) pouring out the fixing solution, adding sterile water (extracting RNA, and then using DEPC treated water) to soak and wash for 3 times, 5min each time;
3) preparing fixed corn root tip tissue protoplasts;
3.1) putting the cleaned fixed tissue into the enzymolysis liquid, putting the fixed tissue into a shaking table, setting the temperature at 35 ℃, rotating speed at 55r/min, carrying out enzymolysis for 5h, and carrying out dark treatment;
the preparation of the enzymolysis liquid comprises the following steps: KH2PO4:27.2mg/L, KNO3:101.0mg/L, CaCl 2.2H2O: 1480mg/L, MgSO 4.7H 2O:246mg/L, CuSO 4.5H 2O:0.025mg/L, KI: 0.16mg/L, mannitol: 14%, cellulase: 2.8%, eductase: 0.6 percent and the balance of hydrosolvent, the PH is adjusted to 5.8 after constant volume, and the filtration and sterilization are carried out at 0.45 mu m, and the mixture is prepared at present;
3.2) after the enzymolysis is finished, sucking the enzymolysis mixed solution by a Pasteur straw, filtering by a nylon mesh screen with the thickness of 0.45 mu m, taking the filtrate, centrifuging for 10min at 1000r/min in a centrifugal tube, and discarding the supernatant; adding 4ml of cell suspension, centrifuging at 1000r/min for 10min, and removing supernatant; repeating the above step 2 times, adding 1ml of cell suspension to the final pellet;
the cell suspension was prepared as follows: KH2PO4:27.2mg/L, KNO3:101.0mg/L, CaCl 2.2H2O: 1480mg/L, MgSO 4.7H 2O:246mg/L, CuSO 4.5H 2O:0.025mg/L, KI: 0.16mg/L, mannitol: 14 percent, the balance being water solvent, filtering and sterilizing at 0.45 mu m, and preparing the traditional Chinese medicine on site;
3.3) protoplast purification: and (3) lightly spreading the zymolyzed protoplast in 80% sucrose solution, centrifuging at 1300r/min for 20min, and purifying the purified protoplast as shown in figure 2.
4) Nucleic acid extraction
4.1) DNA extraction:
4.1.1) centrifuging the purified protoplast at 3000rpm/min for 10min, removing the supernatant, adding 750 μ l CTAB extract preheated at 65 ℃ and 10 μ l mercaptoethanol; keeping the temperature at 65 ℃ for more than 30min, and slightly shaking for 5 times every 10min in the heat preservation process;
4.1.2) taking out the centrifuge tube, cooling to room temperature, adding equal volume of phenol: chloroform: isoamyl alcohol (25:24:1), namely 750 mul, is shaken gently for 5min,12000rpm/min and centrifuged for 10min at 4 ℃;
4.1.3) transfer 600. mu.l of supernatant into a new centrifuge tube, add an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1), namely 600 mul, is shaken gently for 5min,12000rpm/min, centrifuged for 10min at 4 ℃;
4.1.4) taking 400 mul of supernatant again to a new centrifuge tube, adding 0.6 times volume of pre-cooled isopropanol, and standing for more than 30min at room temperature; centrifuging at 12000rpm/min for 2min, discarding the supernatant, washing with 70% ethanol, centrifuging at 12000rpm/min for 2min, and discarding the supernatant;
4.1.5) standing with the cover open, completely drying the residual alcohol at room temperature, adding 30 μ lddH2O, dissolving DNA in water bath at 65 ℃ for 10min, and preserving at-20 ℃.
4.2) RNA extraction;
4.2.1) centrifuging the purified protoplast at 3000rpm/min for 10min, removing the supernatant, adding 1000. mu.l TRNzol lysate, standing at 30 deg.C for 5min,12000rpm/min, and centrifuging at 4 deg.C for 10 min;
4.2.2) taking 800 mul of supernatant fluid to a new centrifuge tube, adding 200 mul of chloroform, reversing and mixing evenly for 2min, placing at room temperature for 3min,12000rpm/min, centrifuging at 4 ℃ for 15min, taking 650 mul of supernatant fluid) to a new centrifuge tube, adding isopropanol with the same volume to the new centrifuge tube, mixing evenly, and placing at room temperature for 30 min; centrifuging at 12000rpm/min at 4 deg.C for 10min, and removing supernatant; adding 1000 μ l of 75% ethanol (RNase-Free ddH2O) to wash the precipitate; centrifuging at 12000rpm/min at 4 deg.C for 3min, pouring out the liquid, centrifuging the rest liquid for a short time, and sucking out with a gun head; standing at room temperature for 3min, adding 30 μ l RNase-Free ddH2O 30, repeatedly beating, and mixing;
5) detecting the quality of nucleic acid;
5.1) nucleic acid concentration detection: with NanoDropTMThe samples obtained in three replicates were tested for DNA and RNA yields, 260nm and 280nm UV absorbance (OD260 and OD280) and OD260/OD280 in a model 2000 spectrophotometer, respectively, and the results are given in Table 2;
table 2 shows the results of the detection of the concentration of nucleic acid according to the present invention
5.2) nucleic acid integrity testing:
the extracted nucleic acid products were subjected to gel electrophoresis using 1% agarose, and image analysis was performed using a gel imager of the GelDoc XR System type, and the results are shown in FIGS. 3 and 4.
DNA amplification: according to known maize gene ASPAT1.2, TUB, sh and zein sequences published in NCBI library, primers 1, 2, 3 and 4 with target fragment lengths of about 200 bp, 400 bp, 600 bp and 800bp are respectively designed by using online software Primer3(http:// Primer3.ut. ee /), and are synthesized by Shanghai biological engineering Co., Ltd; selecting the four genes to carry out PCR detection by taking the extracted DNA as a template; reaction system 25 u L, DNA 100ng,2 x Premix Taq12.5 u L, primer each 0.15mmol.L-1, ddH2O6.5 u L; PCR conditions were 95 ℃ for 5 min; 40 cycles of 95 ℃ for 40s, 50-58 ℃ for 40s, and 72 ℃ for 50 s; extending for 5min at 72 ℃; storing at 4 deg.C; the PCR amplification products were subjected to gel electrophoresis using 1% agarose, and image analysis was performed using a GelDoc XR System type gel imager, and the results are shown in FIG. 5.
RNA amplification: primers 1, 5, 6, 7 were designed using online software Primer3(http:// Primer3.ut. ee /) based on the mRNA sequences of known maize genes ASPAT1.2, TUB, as published in NCBI's library and synthesized by Shanghai Bioengineering, Inc. The 1 st strand cDNA synthesis was performed according to The Instructions (TIANGEN) of FastQuant RT Kit (with gDNase). And carrying out PCR detection on genes with different lengths by taking the reverse transcribed cDNA as a template. The reaction system is 25. mu.L, cDNA3000ng,2 XPromix Taq 12.5. mu.L, upstream and downstream primers 0.15mmol L-1, ddH2O 6.5.5. mu.L each. Reaction program, 95 ℃ for 5 min; 40 cycles of 95 ℃ for 40s, 50-57.5 ℃ for 40s, and 72 ℃ for 50 s; 5min at 72 ℃; storing at 4 ℃. The PCR amplification products were subjected to gel electrophoresis using 1% agarose, and image processing was performed using a GelDoc XR System type gel imaging System, and the results are shown in FIG. 6.
FIGS. 1 and 2 illustrate that protoplasts obtained by enzymatic digestion of fixed tissue retain their original form in the tissue and do not have the property of imbibition.
The method of the second embodiment of the invention is as follows:
1) obtaining young root tip tissues of corn;
selecting full and healthy corn seeds, soaking the corn seeds for 4 hours in ddH2O, after the seeds are fully swelled, intensively placing the seeds on a tray paved with 4 layers of wet gauze, covering a towel on the seeds to keep moisture, placing the seeds in a constant-temperature incubator at 28 ℃ for culturing for 5 days, and cutting young root tips of about 3cm at the front end for fixation;
2) tissue fixation;
2.1) weighing 1.8g of collected root tips, longitudinally cutting the root tips firstly, then transversely cutting the root tips into small sections with the length of 1.2cm, soaking the small sections in a fixing solution, and fixing the small sections at room temperature for 1.2 h; carnoy 2 is selected as stationary liquid, wherein ethanol: glacial acetic acid: chloroform 6:1:3, Methacarn, wherein methanol: glacial acetic acid: chloroform 6:1: 3; fixing the corn root tip tissue;
2.2) pouring out the fixing solution, adding sterile water (extracting RNA, and then using DEPC treated water) to soak and wash for 3 times, 5min each time;
3) preparing fixed corn root tip tissue protoplasts;
3.1) putting the cleaned fixed tissue into the enzymolysis liquid, putting the fixed tissue into a shaking table, setting the temperature at 30 ℃, setting the rotating speed at 60r/min, carrying out enzymolysis for 6h, and carrying out dark treatment; the enzymolysis liquid is prepared from KH2PO4, 27.2mg/L, KNO3, 101.0mg/L, CaCl 2.2H2O, 1480mg/L, MgSO 4.7H2O, 246mg/L, CuSO 4.5H2O, 0.025mg/L, KI: 0.16mg/L, mannitol: 14%, cellulase: 2.8%, eductase: 0.6 percent and the balance of hydrosolvent, the PH is adjusted to 5.8 after constant volume, and the filtration and sterilization are carried out at 0.45 mu m, and the mixture is prepared at present;
3.2) after the enzymolysis is finished, sucking the enzymolysis mixed solution by a Pasteur straw, filtering by a nylon mesh screen with the diameter of 0.45 mu m, taking the filtrate, centrifuging for 12min at 1000r/min in a centrifugal tube, and discarding the supernatant; adding 3ml of cell suspension, centrifuging at 1200r/min for 8min, and removing supernatant; repeating the above step 2 times, adding 1ml of cell suspension to the final pellet;
the cell suspension is prepared from KH2PO4, 27.2mg/L, KNO3, 101.0mg/L, CaCl 2.2H2O, 1480mg/L, MgSO 4.7H2O, 246mg/L, CuSO 4.5H2O, 0.025mg/L, KI: 0.16mg/L, mannitol: 14 percent, the balance being water solvent, filtering and sterilizing at 0.45 mu m, and preparing the traditional Chinese medicine on site;
3.3) purifying the protoplast; gently spreading the protoplast after enzymolysis in 75% sucrose solution, and centrifuging at 1200r/min for 25min for purification;
4) nucleic acid extraction
4.1) DNA extraction:
4.1.1 purified protoplasts were centrifuged at 2500rpm/min for 12min, the supernatant was removed, and 800. mu.l of preheated CTAB extract at 60 ℃ and 12. mu.l of mercaptoethanol were added; keeping the temperature at 70 ℃ for more than 30min, and slightly shaking for 3 times every 10min in the heat preservation process;
4.1.2) taking out the centrifuge tube, cooling to room temperature, adding equal volume of phenol: chloroform: isoamyl alcohol (25:24:1), namely 800 mul, is shaken gently for 3min,12000rpm/min, centrifuged for 12min at 4 ℃;
4.1.3) transfer 600. mu.l of supernatant into a new centrifuge tube, add an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1), namely 600 mul, is gently shaken for 3min and centrifuged at 12000rpm/min at 4 ℃ for 12 min;
4.1.4) taking 400 mul of supernatant again to a new centrifuge tube, adding 0.6 times volume of pre-cooled isopropanol, and standing for more than 30min at room temperature; centrifuging at 12000rpm/min for 2min, discarding the supernatant, washing with 65% ethanol, centrifuging at 14000rpm/min for 2min, and discarding the supernatant;
4.1.5) opening the cover and standing, completely drying the residual alcohol at room temperature, adding 30 mu lddH2O, dissolving DNA in water bath at 65 ℃ for 5-10min, and preserving at-20 ℃;
4.2) RNA extraction:
4.2.1) centrifuging the purified protoplast at 3000rpm/min for 10min, removing the supernatant, adding 800. mu.l TRNzol lysate, standing at 35 deg.C for 5min, 14000rpm/min, centrifuging at 4 deg.C for 10 min;
4.2.2) taking 750 mul of supernatant fluid to a new centrifuge tube, adding 200 mul of chloroform, reversing and mixing evenly for 2min, and standing for 3min at room temperature; centrifuging at 12000rpm/min at 4 deg.C for 15min, collecting supernatant 600 μ l in a new centrifuge tube, adding isopropanol with equal volume, mixing, and standing at room temperature for 30 min; 12000rpm/min,4 ℃, centrifuging for 10min, removing supernatant, adding 1000 mul 75% ethanol (RNase-Free ddH2O) to wash and precipitate 14000rpm/min, centrifuging for 3min at 4 ℃, pouring out liquid, centrifuging the residual small amount of liquid for a short time, sucking out by a gun head, standing for 3min at room temperature, adding RNase-Free ddH2O 30 mul, repeatedly blowing and beating, and uniformly mixing.
5) Detecting the quality of nucleic acid;
5.1) nucleic acid concentration detection: with NanoDropTMRespectively measuring DNA and RNA yields, 260nm and 280nm ultraviolet absorption values OD260 and OD280 and OD260/OD280 of samples obtained by three times of repetition by using a 2000 type spectrophotometer;
5.2) nucleic acid integrity testing;
performing gel electrophoresis on the extracted nucleic acid product by using 1% agarose, and performing image analysis by using a GelDoc XR System type gel imager;
DNA amplification: according to known maize gene ASPAT1.2, TUB, sh and zein sequences published in NCBI library, primers 1, 2, 3 and 4 with target fragment lengths of about 200 bp, 400 bp, 600 bp and 800bp are respectively designed by using online software Primer3(http:// Primer3.ut. ee /), and are synthesized by Shanghai biological engineering Co., Ltd; the extracted DNA is used as a template, and the four genes are selected for PCR detection. Reaction system 25 u L, DNA 100ng,2 x Premix Taq12.5 u L, primer each 0.15mmol.L-1, ddH2O6.5 u L; PCR conditions of 95 ℃, 5min, 95 ℃,40 s, 50-58 ℃,40 s,72 ℃,50 s and 40 cycles; extending for 5min at 72 ℃; storing at 4 ℃, performing gel electrophoresis on the PCR amplification product by using 1% agarose, and analyzing images by using a GelDoc XR System type gel imager;
RNA amplification: according to the mRNA sequences of known maize genes ASPAT1.2, TUB and as published in NCBI library, primers 1, 5, 6 and 7 are designed by using online software Primer3(http:// Primer3.ut. ee /), and synthesized by Shanghai Bioengineering, Inc.; cDNA 1 st chain synthesis was performed according to the (FastQuant RT Kit (with gDNase)) instructions (TIANGEN); carrying out PCR detection on genes with different lengths by taking the reverse transcribed cDNA as a template; the reaction system is 25 mu L, cDNA3000ng,2 XPremix Taq12.5 mu L, the upstream and downstream primers are 0.15mmol L-1 and ddH2O 6.5.5 mu L respectively; the reaction procedure comprises the steps of 95 ℃, 5min, 95 ℃,40 s, 50-57.5 ℃,40 s,72 ℃,50 s and 40 cycles; 72 ℃ for 5 min; the PCR amplification product was stored at 4 ℃ and subjected to gel electrophoresis using 1% agarose, and image processing using a GelDoc XR System type gel imaging split System.
The method of the third embodiment of the invention is as follows:
1) obtaining young root tip tissues of corn;
selecting full and healthy corn seeds, soaking the corn seeds for 6 hours in ddH2O, after the seeds are fully swelled, intensively placing the seeds on a tray paved with 4 layers of wet gauze, covering a towel on the seeds to keep moisture, placing the seeds in a constant-temperature incubator at 36 ℃ for culturing for 4 days, and cutting young root tips of about 3cm at the front end for fixation;
2) tissue fixation;
2.1) weighing 2.2g of collected root tips, longitudinally cutting the root tips firstly, then transversely cutting the root tips into small sections with the length of 0.8cm, soaking the small sections in a fixing solution, and fixing the small sections at room temperature for 0.8 h; carnoy 2 is selected as stationary liquid, wherein ethanol: glacial acetic acid: chloroform 6:1:3, Methacarn, wherein methanol: glacial acetic acid: chloroform 6:1: 3; fixing the corn root tip tissue;
2.2) pouring out the fixing solution, adding sterile water (extracting RNA, and then using DEPC treated water) to soak and wash for 3 times, 5min each time;
3) preparing fixed corn root tip tissue protoplasts;
3.1) putting the cleaned fixed tissue into the enzymolysis liquid, putting the fixed tissue into a shaking table, setting the temperature at 40 ℃, setting the rotating speed at 50r/min, carrying out enzymolysis for 4h, and carrying out dark treatment; the enzymolysis liquid is prepared from KH2PO4, 27.2mg/L, KNO3, 101.0mg/L, CaCl 2.2H2O, 1480mg/L, MgSO 4.7H2O, 246mg/L, CuSO 4.5H2O, 0.025mg/L, KI: 0.16mg/L, mannitol: 14%, cellulase: 2.8%, eductase: 0.6 percent and the balance of hydrosolvent, the PH is adjusted to 5.8 after constant volume, and the filtration and sterilization are carried out at 0.45 mu m, and the mixture is prepared at present;
3.2) after the enzymolysis is finished, sucking the enzymolysis mixed solution by a Pasteur straw, filtering by a nylon mesh screen with the diameter of 0.45 mu m, taking the filtrate, centrifuging for 8min at the speed of 1200r/min in a centrifugal tube, and removing the supernatant; adding 4ml of cell suspension, centrifuging at 1000r/min for 12min, and removing supernatant; repeating the above step 2 times, adding 1ml of cell suspension to the final pellet;
the cell suspension is prepared from KH2PO4, 27.2mg/L, KNO3, 101.0mg/L, CaCl 2.2H2O, 1480mg/L, MgSO 4.7H2O, 246mg/L, CuSO 4.5H2O, 0.025mg/L, KI: 0.16mg/L, mannitol: 14 percent, the balance being water solvent, filtering and sterilizing at 0.45 mu m, and preparing the traditional Chinese medicine on site;
3.3) purifying the protoplast; gently spreading the protoplast after enzymolysis in 90% sucrose solution, and centrifuging at 1400r/min for 15min for purification;
4) nucleic acid extraction
4.1) DNA extraction:
4.1.1 after purification of protoplast 3500rpm/min centrifugal 8min, removing the supernatant, adding 700 u l, 70 degrees C preheating CTAB extract and 8 u l mercaptoethanol; keeping the temperature at 60 ℃ for more than 30min, and shaking gently for 4 times every 10min in the heat preservation process;
4.1.2) taking out the centrifuge tube, cooling to room temperature, adding equal volume of phenol: chloroform: isoamyl alcohol (25:24:1), 700. mu.l, was shaken gently for 4min,14000rpm/min, centrifuged at 4 ℃ for 8 min;
4.1.3) transfer 600. mu.l of supernatant into a new centrifuge tube, add equal volume of phenol: chloroform: isoamyl alcohol (25:24:1), 600 μ l, was gently shaken for 4min, centrifuged at 14000rpm/min for 8min at 4 ℃;
4.1.4) taking 400 mul of supernatant again to a new centrifuge tube, adding 0.6 times volume of pre-cooled isopropanol, and standing for more than 30min at room temperature; 14000rpm/min, centrifuging for 2min, discarding the supernatant, washing with 75% ethanol, centrifuging at 12000rpm/min for 2min, discarding the supernatant;
4.1.5) opening the cover and standing, completely drying the residual alcohol at room temperature, adding 30 mu lddH2O, dissolving DNA in water bath at 65 ℃ for 5-10min, and preserving at-20 ℃;
4.2) RNA extraction:
4.2.1) centrifuging the purified protoplast for 10min at 3500rpm/min, removing the supernatant, adding 1200. mu.l TRNzol lysate, standing at 25 deg.C for 5min,12000rpm/min, and centrifuging at 4 deg.C for 10 min;
4.2.2) taking 850 mul of supernatant fluid to a new centrifuge tube, adding 200 mul of chloroform, reversing and mixing evenly for 2min, and standing for 3min at room temperature; 14000rpm/min, centrifuging at 4 ℃ for 15min, taking 700 mu l of supernatant liquid to a new centrifugal tube, adding isopropanol with the same volume, mixing uniformly, and standing at room temperature for 30 min; 14000rpm/min,4 ℃, centrifuging for 10min, removing supernatant, adding 1000 mul 75% ethanol (RNase-Free ddH2O), washing and precipitating at 12000rpm/min, centrifuging for 3min at 4 ℃, pouring out liquid, centrifuging the residual liquid for a short time, sucking out with a gun head, standing for 3min at room temperature, adding RNase-Free ddH2O 30 mul, repeatedly blowing and beating, and mixing evenly.
5) Detecting the quality of nucleic acid;
5.1) nucleic acid concentration detection: with NanoDropTMRespectively measuring DNA and RNA yields, 260nm and 280nm ultraviolet absorption values OD260 and OD280 and OD260/OD280 of samples obtained by three times of repetition by using a 2000 type spectrophotometer;
5.2) nucleic acid integrity testing;
performing gel electrophoresis on the extracted nucleic acid product by using 1% agarose, and performing image analysis by using a GelDoc XR System type gel imager;
DNA amplification: according to known maize gene ASPAT1.2, TUB, sh and zein sequences published in NCBI library, primers 1, 2, 3 and 4 with target fragment lengths of about 200 bp, 400 bp, 600 bp and 800bp are respectively designed by using online software Primer3(http:// Primer3.ut. ee /), and are synthesized by Shanghai biological engineering Co., Ltd; selecting the four genes to carry out PCR detection by taking the extracted DNA as a template; reaction system 25 u L, DNA 100ng,2 x Premix Taq12.5 u L, primer each 0.15mmol.L-1, ddH2O6.5 u L; PCR conditions of 95 ℃, 5min, 95 ℃,40 s, 50-58 ℃,40 s,72 ℃,50 s and 40 cycles; extending for 5min at 72 ℃; storing at 4 ℃, performing gel electrophoresis on the PCR amplification product by using 1% agarose, and analyzing images by using a GelDoc XR System type gel imager;
RNA amplification: according to the mRNA sequences of known maize genes ASPAT1.2, TUB and as published in NCBI library, primers 1, 5, 6 and 7 are designed by using online software Primer3(http:// Primer3.ut. ee /), and synthesized by Shanghai Bioengineering, Inc.; cDNA 1 st chain synthesis was performed according to the (FastQuant RT Kit (with gDNase)) instructions (TIANGEN); carrying out PCR detection on genes with different lengths by taking the reverse transcribed cDNA as a template; the reaction system is 25 mu L, cDNA3000ng,2 XPremix Taq12.5 mu L, the upstream and downstream primers are 0.15mmol L-1 and ddH2O 6.5.5 mu L respectively; the reaction procedure comprises the steps of 95 ℃, 5min, 95 ℃,40 s, 50-57.5 ℃,40 s,72 ℃,50 s and 40 cycles; 72 ℃ for 5 min; the PCR amplification product was stored at 4 ℃ and subjected to gel electrophoresis using 1% agarose, and image processing using a GelDoc XR System type gel imaging split System.
The above embodiments are only specific embodiments disclosed in the present invention, but the scope of the present invention is not limited thereto, and the scope of the present invention disclosed in the present invention should be subject to the scope of the claims.
Claims (6)
1. A method for extracting protoplast nucleic acid from fixed tissue is characterized in that: the method comprises the following steps:
1) obtaining young root tip tissues of corn;
selecting full and healthy corn seeds, soaking the corn seeds for 4-6 hours in ddH2O, after the seeds are fully swelled, intensively placing the seeds on a tray paved with 4 layers of wet gauze, covering a towel on the seeds to keep moisture, placing the seeds in a constant-temperature incubator at 28-36 ℃ for culturing for 4-5 days, and cutting young root tips with the front end of about 3cm for fixation;
2) tissue fixation;
2.1) weighing 1.8-2.2g of collected root tips, longitudinally cutting the root tips firstly, then transversely cutting the root tips into small sections with the length of 0.8-1.2cm, soaking the small sections in a fixing solution, and fixing the small sections at room temperature for 0.8-1.2 h;
2.2) pouring out the fixing solution, adding sterile water (extracting RNA, and then using DEPC treated water) to soak and wash for 3 times, 5min each time;
3) preparing fixed corn root tip tissue protoplasts;
3.1) putting the cleaned fixed tissue into the enzymolysis liquid, putting the fixed tissue into a shaking table, setting the temperature at 30-40 ℃, setting the rotating speed at 50-60r/min, carrying out enzymolysis for 4-6h, and carrying out dark treatment;
3.2) after the enzymolysis is finished, sucking the enzymolysis mixed solution by a Pasteur straw, filtering by a nylon mesh screen with the thickness of 0.45 mu m, taking the filtrate, centrifuging for 8-12min in a centrifugal tube at the speed of 1000-; adding 3-4 ml of cell suspension, centrifuging for 8-12min at 1000-; repeating the above step 2 times, adding 1ml of cell suspension to the final pellet;
3.3) purifying the protoplast; and gently spreading the protoplast after enzymolysis in 75-90% sucrose solution, and centrifuging at 1200-1400r/min for 15-25min for purification.
4) Nucleic acid extraction
4.1) DNA extraction:
4.1.1 after purification, the protoplast 2500-; keeping the temperature at 60-70 ℃ for more than 30min, and slightly shaking for 3-5 times every 10min in the heat preservation process;
4.1.2) taking out the centrifuge tube, cooling to room temperature, adding equal volume of phenol: chloroform: isoamyl alcohol with the volume ratio of 25:24:1, namely 700-;
4.1.3) transfer 600. mu.l of supernatant into a new centrifuge tube, add equal volume of phenol: chloroform: isoamyl alcohol with the volume ratio of 25:24:1, namely 600 mul, is gently shaken for 3-5min, and centrifuged for 8-12min at 12000-14000rpm/min and 4 ℃;
4.1.4) taking 400 mul of supernatant again to a new centrifuge tube, adding 0.6 times volume of pre-cooled isopropanol, and standing for more than 30min at room temperature; 12000-;
4.1.5) opening the cover and standing, completely drying the residual alcohol at room temperature, adding 30 mu lddH2O, dissolving DNA in water bath at 65 ℃ for 5-10min, and preserving at-20 ℃;
4.2) RNA extraction:
4.2.1) centrifuging the purified protoplast at 3500rpm/min for 10min, removing the supernatant, adding 800-;
4.2.2) taking 850 mul of supernatant fluid 750-; 12000-; 12000 plus 14000rpm/min,4 ℃, centrifugation for 10min, supernatant removal, 1000 mul of 75 percent ethanol (RNase-Free ddH2O) washing precipitation 12000 plus 14000rpm/min,4 ℃, centrifugation for 3min, liquid pouring out, sucking out the residual small amount of liquid by a gun head after short centrifugation, standing for 3min at room temperature, adding RNase-Free dH2O 30 mul, repeatedly blowing and evenly mixing.
2. The method for extracting protoplast nucleic acid from fixed tissue according to claim 1, wherein: carnoy 2 is selected as the stationary liquid in the step 2.1), wherein ethanol: glacial acetic acid: chloroform 6:1:3, Methacarn, wherein methanol: glacial acetic acid: chloroform 6:1: 3; and (5) fixing the corn root tip tissue.
3. The method for fixed tissue protoplast nucleic acid extraction as recited in claim 2, wherein: the enzymolysis liquid in the step 3.1) is prepared into KH2PO4:27.2mg/L, KNO3:101.0mg/L, CaCl 2.2H2O: 1480mg/L, MgSO 4.7H 2O:246mg/L, CuSO 4.5H 2O:0.025mg/L, KI: 0.16mg/L, mannitol: 14%, cellulase: 2.8%, eductase: 0.6 percent and the balance of hydrosolvent, the PH value is adjusted to 5.8 after constant volume, and the filtration and sterilization are carried out at 0.45 mu m, and the preparation is prepared at present.
4. The method for fixed tissue protoplast nucleic acid extraction as recited in claim 3, wherein: the cell suspension in the step 3.2) is prepared into KH2PO4:27.2mg/L, KNO3:101.0mg/L, CaCl 2.2H2O: 1480mg/L, MgSO 4.7H 2O:246mg/L, CuSO 4.5H 2O:0.025mg/L, KI: 0.16mg/L, mannitol: 14 percent and the balance of water solvent, and is filtered and sterilized by 0.45 mu m, and the preparation is prepared just before use.
5. The method for fixed tissue protoplast nucleic acid extraction as claimed in any one of claims 1 to 4, wherein: the step 4) further comprises the step 5) of detecting the quality of the nucleic acid;
5.1) nucleic acid concentration detection: with NanoDropTMRespectively measuring DNA and RNA yields, 260nm and 280nm ultraviolet absorption values OD260 and OD280 and OD260/OD280 of samples obtained by three times of repetition by using a 2000 type spectrophotometer;
5.2) nucleic acid integrity testing;
the extracted nucleic acid products were subjected to gel electrophoresis with 1% agarose and image analysis by a gel imager of the GelDoc XR System type.
6. The method for fixed tissue protoplast nucleic acid extraction as recited in claim 5, wherein: the specific steps of the step 5.2) are as follows:
DNA amplification: according to known maize gene ASPAT1.2, TUB, sh and zein sequences published in NCBI library, primers 1, 2, 3 and 4 with target fragment lengths of 200, 400, 600 and 800bp are respectively designed by utilizing online software Primer3 and synthesized by Shanghai biological engineering Limited company; selecting the four genes to carry out PCR detection by taking the extracted DNA as a template; reaction system 25 u L, DNA 100ng,2 x Premix Taq12.5 u L, primer each 0.15mmol.L-1, ddH2O6.5 u L; PCR conditions of 95 ℃, 5min, 95 ℃,40 s, 50-58 ℃,40 s,72 ℃,50 s and 40 cycles; extending for 5min at 72 ℃; storing at 4 ℃, performing gel electrophoresis on the PCR amplification product by using 1% agarose, and analyzing images by using a GelDoc XR System type gel imager;
RNA amplification: according to the mRNA sequences of known corn genes ASPAT1.2, TUB and as published in NCBI library, primers 1, 5, 6 and 7 are designed by using online software Primer3 and synthesized by Shanghai biological engineering GmbH; strand 1 cDNA synthesis was performed according to TIANGEN instructions; carrying out PCR detection on genes with different lengths by taking the reverse transcribed cDNA as a template; the reaction system is 25 mu L, cDNA3000ng,2 XPremix Taq12.5 mu L, the upstream and downstream primers are respectively 0.15mmol L-1 and ddH2O 6.5.5 mu L; the reaction procedure comprises the steps of 95 ℃, 5min, 95 ℃,40 s, 50-57.5 ℃,40 s,72 ℃,50 s and 40 cycles; 72 ℃ for 5 min; the PCR amplification product was stored at 4 ℃ and subjected to gel electrophoresis using 1% agarose, and image processing using a GelDoc XR System type gel imaging split System.
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| US20210285852A1 (en) * | 2020-03-10 | 2021-09-16 | The Board Of Trustees Of The Leland Stanford Junior University | Methods to isolate cells from fixed tissue |
| US11519831B2 (en) * | 2020-03-10 | 2022-12-06 | The Board Of Trustees Of The Leland Stanford Junior University | Methods to isolate cells from fixed tissue |
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