Nano RNAi preparation and its preparation method and application
A kind of nano-technology and preparation technology, applied in the field of genetic engineering, can solve the problems of poor stability and systemicity, and achieve good results, environmental friendliness, and improved stability
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Embodiment 1
[0037] A preparation of chitosan of the present invention in conjunction with TMV dsRNA nano RNAi preparation, its preparation method may further comprise the steps:
[0038] (1) Extraction of Tobacco Mosaic Virus RNA: After collecting tobacco leaves infected with TMV, the RNA was extracted by the Trizol method. The specific operation is as follows:
[0039] 1.1. Take 0.1 g of the diseased leaves of fresh plants that have been weighed and place them in a sterilized mortar, pour an appropriate amount of liquid nitrogen into the mortar, and quickly grind the leaves frozen by liquid nitrogen into a relatively uniform powder or liquid , and then quickly transfer the ground powder or liquid into a 1.5 mL RNase-free centrifuge tube.
[0040] 1.2. Quickly draw 1 mL of Trizol into the centrifuge tube, mix the two thoroughly, and place the centrifuge tube at room temperature for 5 minutes.
[0041] 1.3. Place the centrifuge tube at 4°C, 12000 rpm, and centrifuge for 10 min.
[0042]...
Embodiment 2
[0087] The mensuration of the stability of the nano-sized RNAi preparation of embodiment 1, its assay method comprises the following steps:
[0088] 1. Take 5 μL of the nano-sized RNAi preparation in Example 1 and the dsRNA synthesized in vitro and place them in RNase-free PCR tubes, 45 tubes each; seal the RNase-free PCR tubes with Parafilm to avoid liquid evaporation, and centrifuge briefly;
[0089] 2. Divide the 45 tubes into three parts and place them on the PCR board in turn, and place them at 4°C, 25°C, and 37°C for 30 days;
[0090] 3. Carry out agarose gel electrophoresis detection every 2 days. During electrophoresis, take 3 μL loading buffer, 3 μL ethidium bromide fluorescent dye and 3 μL sample, mix well, and then point them into the sample wells respectively. After electrophoresis at 130 V for 25 min , observe and save the electropherogram (see image 3 B. Figure 4 B. Figure 5 B);
[0091] 4. At the same time, measure the amount of dsRNA with NanoDrop 2000c ...
Embodiment 3
[0095] A kind of application of the nano RNAi preparation of embodiment 1 in TMV prevention and treatment, specific application method is as follows:
[0096] The half-leaf method was used to study the prevention and treatment of nanomaterials-RNAi preparations on tobacco mosaic virus, and four groups of treatments were designed, (1) clean water was used as the blank control treatment, 1v / v%dsRNA treatment; (2) clear water was used as the treatment Blank control treatment, 1v / v% CS-RNAi preparation treatment; (3) clean water as blank control treatment, 0.1% morpholine hydrochloride treatment; (4) clean water as blank control treatment, CS nanomaterial treatment; each treatment 10 plants, the experiment was repeated 3 times.
[0097] Taking the main vein as the interval, the left part of the leaf is the control group, and the right part of the leaf is the treatment group. The number of dead spots of the preventive and therapeutic effects is the average of the total number of de...
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