Method for preparing inhibitor for reducing tobacco nicotine synthesis by double-stranded RNA interference technology and application thereof
An RNA interference and inhibitor technology, applied in DNA preparation, recombinant DNA technology, botanical equipment and methods, etc., can solve problems such as difficulty in popularization and application, and achieve low cost, broad industrialization prospects, and no residue and environmental pollution. Effect
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Embodiment 1
[0028] 1. Extraction of total RNA from tobacco roots
[0029] The main tobacco variety Yunyan 87 in my country was used as the material. After washing the young roots with sterile water, the total RNA of tobacco was extracted with TRNzol TotalRNA Reagent.
[0030] 1) Take 0.1 g of young tobacco root and grind it sufficiently in liquid nitrogen for 55 seconds, and add 1 mL of TRNzol;
[0031] 2) Place the homogenized sample at 15-30°C for 5 minutes to completely separate the nucleic acid-protein complex;
[0032] 3) Centrifuge at 12000rpm for 10m at 4°C, and take the supernatant;
[0033] 4) Add 0.2mL chloroform, cover the tube cap, shake vigorously for 15s, and place at room temperature for 3min;
[0034] 5) Centrifuge at 4°C, 12000rpm for 10-15m, the sample will be divided into three layers: the yellow organic phase, the middle layer and the upper colorless water phase, RNA is mainly in the water phase, transfer the water phase to a new centrifuge tube;
[0035] 6) Add an ...
Embodiment 2
[0195] application of the inhibitor nornicotine,
[0196] Two days before tobacco topping, the inhibitor containing 30ug / mL dsRNA was sprayed on the root surface of the tobacco in the experimental group before 9 am or after 5 pm, and the application rate was 10mL / plant. The experimental group and the control group each have 4 groups, and each group has 100 tobacco plants, which are harvested and baked after normal maturity, and the nicotine content in the tobacco leaves is sampled to determine the content of nicotine. As a result of the experiment, compared with the control, the nicotine content in the middle leaves decreased by 43.36%, and the nicotine content in the upper leaves decreased by 55.42%, as shown in Table 1.
[0197] Table 1 Nicotine content in tobacco leaves of hairpin dsRNA preparation of PMT gene partial sequence applied by root application (%)
[0198]
[0199] Note: * indicates that the difference between the data in the same column and the control reach...
Embodiment 3
[0201] In this example, 10 mL / plant of the inhibitor containing 30 ug / mL dsRNA was applied to the leaves of tobacco. Others are identical with embodiment 2. According to the experimental results, compared with the control, the nicotine content in the middle leaves decreased by 18.92%, and the nicotine content in the upper leaves decreased by 23.31%.
[0202] Table 2 Nicotine content (%) in tobacco leaves of hairpin dsRNA preparation of PMT gene partial sequence by foliar application
[0203]
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