[0035] 1 Preparation of Southern probes.
[0036] 1.1 Types of probes.
[0037]There are two types, one is "anchor probe", which is used for the first round of screening of the BAC library of wild rice; the other is "defining probe", which is used for further screening of positive clones identified in the first round of screening. The former uses tiller control genes MOC1 , the latter is MOC1 2 genes upstream (from MOC1 About 42kb, one at 53kb) probe and 3 downstream genes (distance from MOC1 Probes at approximately 42kb, one at 52kb, and one at 62kb).
[0038] 1.2 Probe PCR amplification.
[0039] With japonica rice Nipponbare ( Oryzasativa L.ssp. japonica cv. Nipponbare) was used as a template for PCR amplification. The primer sequences are shown in Table 1.
[0040] "Anchor Probe" MOC1 Because of its high GC content (>75%), GC buffer (GCbufferⅠ, containing 5mmol/LMg2+) was used for the amplification of . The system was: LA Taq Enzyme, 0.3 μL; 2×GCbuffer I, 12.5 μL; dNTP, 3 μL; template DNA 100 ng; primer 4 μL; add ddH2O to 25 μL. The amplification program was as follows: 94°C, denaturation for 4 min; (94°C, 30s; 59°C, 30s; 72°C, 1min) 30 cycles; 72°C, extension for 5min; storage at 4°C.
[0041] Amplification of "defining probes" is carried out in a conventional manner. The system is: ordinary Taq Enzyme, 0.25μL; 10×buffer (containing 5mmol/LMg2+), 2.5μL; dNTP, 2μL; template DNA 100ng; primer 2μL; add ddH2O to 25μL. The amplification program was as follows: 94°C, denaturation for 4 min; (94°C, 30s; 58°C, 30s; 72°C, 1min) 30 cycles; 72°C, extension for 5min; storage at 4°C.
[0042] 1.3 PCR amplification product recovery.
[0043] PCR amplification products were recovered using a DNA gel recovery kit.
[0044] 1.4 Vector ligation, transformation and cloning.
[0045] After the recovered DNA was detected by electrophoresis, the recovery quality and recovery rate were connected to the pGEMT-easy vector, MOC1 The ligation system is: 1 μL of ligase, 0.2 μL of T vector, 8 μL of 2×buffer, MOC1 The recovered DNA solution was 6.8 μL, and the total system was 16 μL. Place in a refrigerator at 4°C and ligate overnight.
[0046] Transformation was performed using the electroshock method, following the method described in the "Experimental Guide for Molecular Cloning" (Sambrook and Brassell, 2008). After transformation, restore the culture in SOC medium at 37°C with gentle shaking for 60-90 min, then draw 50-100 μL of the transformed bacterial solution and spread it on the LB plate pre-coated with IPTG and X-gal with a sterile spreader. Clonal propagation was performed overnight in a 37°C incubator.
[0047] Pick the monoclonal leukoplakia, put it into a test tube containing 2 mL of liquid LB medium, and cultivate overnight at 37°C with vigorous shaking at 220 r/min. After that, the plasmid DNA was extracted according to the extraction method of plasmid DNA in "Molecular Cloning Experiment Guide". The instrument used for the determination of the DNA fragment insertion sequence on the T vector was ABI3730.
[0048] For the "limited probe", ligation and transformation were not performed due to the small amount, and only the recovered DNA was stored at -20°C for later use.
[0049] 2BAC library Southern hybridization.
[0050] 2.1 Reagents.
[0051] (1) 50×Denhardt's solution: 5g Ficoll-40, 5g PVP, 5g BSA, add water to 500mL, filter and sterilize and store at -20°C.
[0052] (2) 1×BLOTTO: 5g skim milk powder, 0.02% sodium azide, stored at 4°C.
[0053] (3) Pre-hybridization solution: 6×SSC, 5×Denhardt, 0.5% SDS, 100 mg/mL salmon sperm DNA.
[0054] (4) Hybridization solution: adding denatured probes to the pre-hybridization solution is the hybridization solution.
[0055] (5) 10% (W/V) SDS: Weigh 100g of high-purity SDS into a 1000mL beaker, add about 800mL of deionized water, heat to dissolve at 68°C, and dropwise add concentrated hydrochloric acid to adjust the pH to 7.2. After volume to 1000mL, store at room temperature. 0.5%, 0.1% SDS was diluted with 10% SDS.
[0056] (6) 0.4mol/L NaOH.
[0057] (7) 20×SSC: 3 mol/L NaCl, 0.3 mol/L sodium citrate, adjust pH to 7.0 with 1 mol/L HCl, sterilize by high pressure moist heat for 20 min, and store at room temperature; 2×, 1× and 0.1× SSC: use 20× SSC dilution.
[0058] 2.2 Operation steps.
[0059] (1) Open the molecular hybridization box in advance and adjust the temperature to 68°C.
[0060] (2) Wet the BAC membrane with 6×SSC for 2 min.
[0061] (3) Put the BAC membrane into the hybridization bottle, pour 50 mL of pre-hybridization solution, put it back into the hybridization box, and pre-hybridize at 68°C for 3-12 hours.
[0062] (4) Preparation of isotope-labeled probe: Probe preparation was performed according to the method provided by the random primer labeling kit (Promega). The labeled probe was added to an equal volume of 0.4mol/L NaOH for denaturation for 10~15min.
[0063] (5) Take out the pre-hybridized hybridization bottle, add the labeled denatured probe to the hybridization solution, mix well, and hybridize at the same temperature as the pre-hybridization for 7-12 hours.
[0064] (6) Washing the membrane: Take out the BAC membrane from the hybridization bottle, rinse it with the following solutions in turn, and shake gently: a. At room temperature, 2×SSC/0.5% SDS, 5min; b. At room temperature, 1×SSC/0.1 %SDS, 15min; c. 0.1×SSC/0.1%SDS, 30min~3h at 65℃. In the process of washing the membrane, the hybridization signal on the BAC membrane should be checked with the detector at any time. When there is a signal in some areas of the membrane and no signal in some areas, the water droplets on the membrane should be blotted dry with absorbent paper, and wrapped with plastic wrap. .
[0065] (7) Signal detection: use a phosphorscreen imaging system to temporarily store the hybridization signal on the BAC membrane, scan it with a Typhoon 9200 laser scanning imager (purchased from Amersham), and use ImageQuent software to convert the information image to the corresponding signal on the display pixels, resulting in an accurate image of the original sample. ImageQuent quantifies the difference in signal, the level of which is proportional to the amount of radioactivity in the sample.
[0066] 3BAC library positive clone identification.
[0067] The BAC high-density library membrane used in the present invention adopts the type of 4×4 six Fields. The method recommended by the Genome Institute of the University of Arizona (http://www.genome.arizona.edu/) was used for the identification of positive clones in the BAC library.
[0068] 4BACDNA extraction.
[0069] Basically the same as the extraction method of plasmid DNA, the difference is that when the cells are collected by centrifugation for the first time, the cells are resuspended once with pre-cooled STE, and then the cells are collected by centrifugation in the same way. In addition, an equal volume of isopropanol is generally used for precipitation of BACDNA. Composition of STE solution: 10mmol/LTris-HCl (pH8.0), 1mmol/LEDTA (pH8.0), 0.1mol/LNaCl.
[0070] 5BACDNA digestion and membrane transfer.
[0071] 5.1BACDNA digestion and electrophoresis.
[0072] Using a 20 μL system: HindⅢ Endonuclease 0.3 μL, 10× buffer ② 2 μL, BACDNA 100~200 ng, add ddH2O to 20 μL, shake and mix, and digest overnight at 37°C. After the digestion is completed, electrophoresis in a 1% agarose gel at a voltage of 1V/cm for 20-36h (including DNA molecular mass standards).
[0073] 5.2 Transfer film.
[0074] 5.2.1 Reagents.
[0075] Alkaline transfer buffer: 0.4 mol/L NaOH, 1 mol/L NaCl. For the preparation methods of 10mol/L NaOH and 5mol/L NaCl mother solutions, see "Molecular Cloning Experiment Guide" (Sambrook and Brassell, 2008).
[0076] 5.2.2 Operation steps.
[0077] (1) Place the gel after electrophoresis in ethidium bromide (EB) for 5 minutes and then take a picture.
[0078] (2) Trim the edge of the gel with a knife (to facilitate the removal of air bubbles when transferring the membrane) and cut off a corner of the gel as a marker, place it in the transfer buffer and shake it gently for 15 minutes, repeating once.
[0079] (3) Prepare the transfer device: wash the appropriate size tray, glass plate for support, etc., rinse three times with ddH2O, and pour an appropriate amount of transfer buffer; HybondN+ nylon membranes (purchased from Amersham) were cut to the appropriate gel size for use.
[0080] (4) Assemble the transfer device: put the glass plate on the tray, cover it with a layer of moist absorbent paper, invert the denatured gel so that the original bottom surface is upward and gently place it on the moist absorbent paper, and then apply the gel to the gel. Lay 2 layers of absorbent paper on top, and finally spread a stack of paper towels with a thickness of 5~8cm on it, and press it with a weight of about 400g on the top, and transfer it at room temperature for 8~12 hours. Note: The generation of air bubbles should be avoided during the entire assembly process, otherwise the transfer effect will be affected.
[0081] (5) Fixation: After the transfer is completed, the film is removed and placed in an oven at 80°C for 2 hours. After that, write the serial number on the membrane and store it in a 4°C refrigerator for later use.
