Chrysanthemum drought-tolerant gene and application thereof
A chrysanthemum and gene technology, applied in the application of OtMYB gene in improving the drought resistance of chrysanthemum, in the field of chrysanthemum drought resistance related gene OtMYB, can solve the problem of complex genetic background of chrysanthemum
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Embodiment 1
[0021] Cloning of embodiment 1 OtMYB gene
[0022] 1. Build Target Groups
[0023] The PEG solution was used to stress the tissue-cultured seedlings of Chrysanthemum chrysanthemum under drought stress, and after sampling, they were quick-frozen in liquid nitrogen and stored at -80°C as a population for drought-resistant gene mapping;
[0024] 2. Extraction of total RNA and isolation of mRNA
[0025] 1) RNA extraction method: take 100 mg of tender material, add liquid nitrogen to fully grind, put it into a 2ml centrifuge tube, add 1ml Trizol reagent, shake vigorously and mix well, let stand at room temperature for 10min; centrifuge at 12000rpm at 4°C for 15min to remove the precipitate; Transfer the supernatant to a new centrifuge tube, add an equal volume of phenol / chloroform / isoamyl alcohol (25:24:1), shake vigorously, and let stand for 10 minutes; centrifuge again at 12,000 rpm at 4°C for 15 minutes; transfer the upper aqueous phase to a new tube Add an equal volume of chl...
Embodiment 2
[0041] The construction of embodiment 2OtMYB expression vector
[0042] 1. Use Escherichia coli DH5a to prepare competent cells, and prepare a colony culture solution with an OD value of 0.6-0.9. Take 3-5 μL of the cloning vector and pBI121 plasmid DNA in competent cells, and transform the recombinant plasmid into E. coli; extract the E. coli plasmid DNA.
[0043] 2. Digestion and recovery of DNA fragments
[0044] The buffer used for plasmid digestion is selected according to the Takara reagent catalog, the buffer provided with the product is selected for single digestion, and the optimal buffer is selected according to the reagent catalog for double digestion. Generally, add at least 1 U of enzyme to 1 μg of plasmid, and incubate at 37°C for 2 hours.
[0045] In this experiment, a kit was used to extract the plasmid, which has a high purity and a good enzyme digestion effect. Generally, it can be cut in 1 hour, and it can be completely digested in 3-4 hours. It is better ...
Embodiment 3
[0075] Example 3 Result and application of expression of drought-resistance-related gene OtMYB
[0076] According to the cloning sequence where the OtMYB gene is located, the expression vector pBI121 and the intermediate cloning vector were respectively digested with restriction enzymes XbaI and SmaI for directional sticky-end-blunt-end ligation, and the genetic transformation method mediated by Agrobacterium EHA105 was used to convert the genome The vector was introduced into the leaf explants of the ground cover chrysanthemum variety 'Wanfen', and finally 5 OtMYB positive plants were obtained.
[0077] Five transgenic lines T1, T2, T3, T4, T5 of ground cover chrysanthemum variety 'Wanfen' and control CK were planted in an incubator. Select 8-10 chrysanthemum seedlings in the true leaf stage for stress treatment, and control water for 4 days under the condition of 30° C. and 30% RH. The 40-day-old chrysanthemum seedlings were selected to be treated with water control for 4 d...
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