A kind of tobacco anti-low temperature stress inducible early flowering gene ntmyb15 and its cloning method and application
A technology of anti-low temperature and tobacco, applied in the field of genetic engineering
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[0087] Preparation of Agrobacterium Infection Solution
[0088] (1) Take the Agrobacterium containing the expression vector stored in the -80°C refrigerator, culture it on a flat plate, and add 50mg / LKan and 50mg / LRif to the LB solid plate;
[0089] (2) Pick a single plaque into 5mL LB liquid medium containing 50mg / LKan and 50mg / LRif, put it in a shaker at 28°C, 200rpm and culture overnight (12h-16h);
[0090] (3) To preserve the strains, add 250ul of sterilized glycerin to 750ul of the bacterial solution, and store in a -80°C refrigerator for later use.
[0091] (4) Shake the bacteria, add 10ml of LB liquid medium, 10ul of Kan (required concentration 50mg / L), 10ul of Rif (required concentration of 50mg / L) and 10ul of bacterial liquid, culture overnight at 28°C and 200rpm (12h-16h).
[0092] (5) When the concentration of the bacterial solution reaches about OD600=1.5, take 2mL of the bacterial solution and add it to the centrifuge tube, and centrifuge at 4000rpm for 5min;
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Embodiment 1
[0098] Low temperature stress can lead to early flowering of tobacco
[0099] Tobacco plants at the 6-7 leaf stage were treated under low temperature stress at 12°C for 14 days, and tobacco plants grown at normal temperature were set as controls, and were treated at 12°C for low temperature stress for 14 days and then moved to normal temperature until flowering was induced by low temperature. experimental conditions. The early flowering phenotype could be reproduced stably, and the flowering time of tobacco treated with low temperature stress was advanced by an average of 15 days (Figure 1).
Embodiment 2
[0101] 1. Tobacco leaf cDNA synthesis
[0102] The total RNA of tobacco leaves was extracted with TRIZOL (Invitrogen, USA) reagent, 1 μg of total RNA of tobacco leaves was quantified in a 1.5ml centrifuge tube, and reverse transcription was performed according to the instructions of Invitrogen’s First Strand cDNA Synthesis Kit to finally obtain cDNA of tobacco leaves.
[0103] 2. NtMYB15 PCR amplification of the RNAi fragment of the gene
[0104] Tobacco leaf cDNA was used as a template, and primers were designed according to the tobacco genome database information, and the NtMYB15 The RNAi carrier fragment of the gene is amplified by PCR to obtain a PCR amplification product.
[0105] Primers are:
[0106] Forward primer: 5'-CCACTTGAAAAAGAGGCTTAAAAAT-3'
[0107] Reverse primer: 5'-TAAAATTTTCAGACGAGTCCATCTC-3'
[0108] The PCR product obtained by amplification was electrophoresed on 0.8% agarose gel, and the gel electrophoresis result was (as figure 2 shown), and a 29...
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