A kind of stem mustard glucosinase gene myr1, stem mustard glucosinase and gene engineering expression method thereof
A glucosinolate and genetic engineering technology, applied in the field of glucosidase, can solve the problems of no extraction, low content of glucosinolate, inability to obtain glucosidase in large quantities and the like
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Embodiment 1
[0044] The extraction of embodiment 1 stem mustard total RNA:
[0045] Add 5 mL Trizol reagent to the centrifuge tube, take 0.5 g stem mustard leaf and quickly grind it into powder in liquid nitrogen, then transfer it to a test tube, mix it thoroughly immediately, and place it at room temperature for 10 min, then place it in a refrigerated centrifuge Centrifuge, set the temperature at 4°C, and rotate at 7200 rpm / min, absorb the upper layer liquid after centrifugation for 25 min; add CHCl to the upper layer liquid 31 mL and 1 mL of 5 mol / L NaCl solution, shake vigorously and place for 3 min, then centrifuge at 4°C, 7200 rpm / min for 25 min, then the sample will be divided into 3 layers, take the supernatant; add 2.5 mL to the supernatant Isopropanol, placed for 10 min, centrifuged again for 20 min, discarded the supernatant, a gelatinous precipitate formed on the bottom side of the centrifuge tube; added 5 mL ethanol to the centrifuge tube to wash the precipitate, centrifuged fo...
Embodiment 2
[0046] Example 2 Gel electrophoresis detection of total RNA
[0047] Add 25 mL of 1×TAE buffer and 0.25 g of agarose to the Erlenmeyer flask, heat it in a microwave oven until the agarose melts, and when it cools down to a temperature that the back of the hand can bear, add 2.5 μL of GoldviewTM nucleic acid dye, mix and pour In the glue board. Take 4 μL of the total RNA extracted and dissolved in Example 1 and mix it with 1 μL of Loading buffer before adding the sample. After electrophoresis at 70mA and 100V for 30 min, observe under ultraviolet light. The results are as follows: figure 1 as shown, figure 1 The labels of the lanes are: R1, R2, R3, the three lanes are extracted RNA, from figure 1 Three bands can be seen, and the brightness ratio of the 28S band to the 18S band is about 2:1, which proves that the proposed total RNA can be used in subsequent experiments.
Embodiment 3
[0048] Example 3 Synthesis of first-strand cDNA
[0049] Use the AMV first-strand cDNA synthesis kit from Shanghai Sangon Bioengineering Co., Ltd. to synthesize the first-strand cDNA. Insert the centrifuge tube into ice and add the following reagents:
[0050] Example 1 Extract 2 μL of dissolved total RNA
[0051] Oligo dt Primer (0.5 μg / μL) 1 μL
[0052] RNase free ddH 2 O to make a final volume of 12 µL
[0053] Centrifuge the above-prepared solution with a total volume of 12 μL at 4000 rpm / min for 2 min, then place the reaction mixture in a water bath at 65 °C for 5 min, then insert it into ice for 30 s, centrifuge for 2 min, and take supernatant.
[0054] Place the tube containing the supernatant on ice and add the following components:
[0055] 5×Reaction buffer 4 μL
[0056] RNase Inhibitor (20M / μL) 1 μL
[0057] DNTP Mix (10 mmol / L) 2 μL
[0058] AMV Reverse Transcriptase (10Μ / μl) 2 μL
[0059] After mixing, centrifuge for 2 min, carry out reverse transcription...
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