RNA interference vector fragment, RNA interference vector and application
A technology of RNA interference and carrier, applied in the field of genetic engineering, can solve problems such as root nodules of legumes that have not yet been seen
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Embodiment 1
[0023] Example 1, Analysis of the expression pattern of GmNNC1 gene in early response to rhizobia infection
[0024] 1) Material acquisition: The material used in the experiment is Williams 82 (W82 for short). The materials are processed according to the following process: soybean seeds are sterilized with 70% alcohol for 30 seconds, sown in a mixed substrate of vermiculite and perlite (3:1) soaked in low-nitrogen nutrient solution, cultivated in a culture room, 16h light / 8h dark , the light intensity is 7000LUX, the temperature is 26°C, and the relative humidity is 70%. Seven days after sowing, each plant was inoculated with Bradyrhizobium USDA110 bacterial solution (OD 600 : 0.08) 30ml, take soybean roots respectively at 0, 1, 3, 6, 12 and 24 hours after inoculation;
[0025] 2) Isolation of mRNA: Use the Trizol method to extract total soybean RNA. ①Put the tissue into a grinder and grind it with liquid nitrogen for 3 times. It is a clear transparent viscous liquid. Place...
Embodiment 2
[0029] Example 2, Analysis of the expression pattern of GmNNC1 at different stages of root nodule development
[0030] 1) Material acquisition: The material used in the experiment was Williams 82 (W82 for short); the material was carried out according to the following process: soybean seeds were sterilized with 70% alcohol for 30 seconds, and sowed in vermiculite perlite soaked in low-nitrogen nutrient solution (3 :1) In the mixed medium, culture in the culture room, 16h light / 8h dark, light intensity 7000LUX, temperature 26°C, relative humidity 70%. Seven days after sowing, each plant was inoculated with Bradyrhizobium USDA110 bacterial solution (OD 600 =0.08) 30ml, soybean root and leaf tissues were collected at 0, 1, 3, 5, and 10 days after inoculation;
[0031]2) Isolation of mRNA: Use the Trizol method to extract soybean total RNA. ①Put the tissue into a grinder and grind it with liquid nitrogen for 3 times, add 50-100 mg of the ground tissue into a 1.5 mL centrifuge tub...
Embodiment 3
[0035] Example 3. Using the RNA interference vector to down-regulate the GmNNC1 gene to cultivate transgenic soybeans with high ability to nodulate and fix nitrogen.
[0036] 1) Extraction of total RNA from functional leaf tissue of Wilimas82 soybean
[0037] The mortar has been treated at 180°C for 8 hours or burned to eliminate RNase contamination; reagents such as chloroform, isopropanol, and ethanol should be freshly opened and uncontaminated; other equipment such as pipette tips, centrifuge tubes and reagents such as ultrapure water , NaAc, were treated with 1‰ DEPC water overnight, then sterilized at 121°C for 30 minutes, and the pipette tip and centrifuge tube were dried at 65°C for later use; Trizol method was used to extract soybean total RNA:
[0038] (1) Take 50mg of material (leaf) and grind it with liquid nitrogen, add 1ml of redzol reagent, after fully homogenizing, inhale the homogenate into a 1.5ml centrifuge tube, and place it at room temperature for 5 minutes...
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