Small RNA (ribonucleic acid) molecule inhibiting toxicity of rice blast fungi and application thereof
A technology of rice blast fungus and rice blast, which is applied in the field of agricultural biology, can solve the problems of no discovery and control of rice blast, and achieve the effects of environmental protection, inhibition of infection, and good stability
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Embodiment 1
[0035] Embodiment 1 The present invention suppresses the source of the small RNA molecule of rice blast fungus toxicity
[0036] (1). The first 19 bases in the small RNA molecule of the present invention are derived from the cDNA coding sequence of MoAP1, and the sequence is shown in SEQ ID No.3. The cDNA coding sequence of MoAP1 was derived from the database of Magnaporthe grisea ( http: / / www.broadinstitute.org / annotation / genome / magnaporthe_grisea / MultiHome.html) Gene numbered MGG_12814 in .
[0037] (2). A total of 19 nucleotide sequences from No. 1081 to 1099 of the cDNA coding sequence of MoAP1 are selected as the first 19 nucleotide sequences of the small RNA molecule of the present invention.
[0038] (3). In order to ensure the stability of the small RNA molecule, the small RNA molecule is designed as dsRNA, and two thymines are added at the 3' end of each strand to stabilize the small RNA structure and form a complete 21-base small RNA molecule. Compare this seq...
Embodiment 2
[0039] Embodiment 2 Preparation of small RNA molecule of the present invention
[0040] The small RNA molecules of the present invention (see the nucleic acid sequences of SEQ ID No.1 and SEQ ID No.2) entrust Shanghai Gemma Pharmaceutical Technology Co., Ltd. to directly synthesize the double strands, and then dry them into dry powder, which is convenient for transportation. Aliquot one EP tube for every 0.005 μmol RNA dry powder. Add 100 μl double-distilled water to every 0.005 μmol RNA dry powder, dissolve and dilute it into a 50 μM working stock solution, and freeze it at -80°C for later use.
[0041] Take 3 μl and dilute to 30 μl to prepare a small RNA solution with a final concentration of 5 μM. Distribute 30 μl of the small RNA solution of the present invention into three 1.5 ml EP tubes, 10 μl in each tube. Place the three EP tubes containing the small RNA solution of the present invention at room temperature for 0, 24, and 48 hours respectively; then use 1% agarose gel...
Embodiment 3
[0042] Embodiment 3 Small RNA molecules of the present invention are fed to rice blast fungus test
[0043] (1) Preparation of a complete nutrient medium (CM for short) for cultivating Magnaporthe grisea. The composition and ratio of the complete nutrient medium are: glucose 10g / L, peptone 2g / L, yeast extract 1g / L, chelatin amino acid 1g / L, NaNO 3 0.6g / L, KCl0.05g / L, MgSO 4 0.05g / L,KH 2 PO 4 0.15g / L, biotin 1mg / L, vitamin B61mg / L, vitamin B11mg / L, riboflavin 1mg / L, β-aminobenzoic acid 1mg / L, niacin 1mg / L, agar 15g / L, plus double Make up to 1L with distilled water; adjust the pH to 6.5 after mixing, and autoclave for later use. Add 100 μl of the 50 μM small RNA working mother solution prepared in Example 2 to 100 ml CM medium to prepare a CM medium containing a final concentration of 50 nM small RNA molecules. Similarly, 200 μl and 400 μl of small RNA working master solution with a concentration of 50 μM were added to 100 ml of CM medium respectively to prepare CM medium w...
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