Chrysanthemum drought-tolerant gene and application thereof
A chrysanthemum and gene technology, applied in the application of OtMYB gene in improving the drought resistance ability 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] Example 10 Cloning of OtMYB gene
[0022] 1. Establish positioning groups
[0023] Tissue cultured seedlings of Taihang chrysanthemum were subjected to drought stress with PEG solution, and then quickly frozen in liquid nitrogen after sampling and stored at -80°C as a drought resistance gene mapping population;
[0024] 2. Extraction of total RNA and separation of mRNA
[0025] 1) RNA extraction method: take 100mg of young material, add liquid nitrogen to fully grind, put into a 2ml centrifuge tube, add 1ml Trizol reagent, vigorously shake and mix, let stand at room temperature for 10min; centrifuge at 12000rpm for 15min at 4℃ 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 12000 rpm at 4°C for 15 minutes; move the upper water phase to the new In the centrifuge tube, add an equal volume of chloroform / isoamyl alcoh...
Embodiment 2
[0041] Example 2 Construction of OtMYB expression vector
[0042] 1. Use E. coli DH5a to prepare competent cells, and prepare a colony culture medium with an OD value of 0.6-0.9. Take 3-5μL of cloning vector and pBI121 plasmid DNA in competent cells, transform the recombinant plasmid into E. coli; extract E. coli plasmid DNA.
[0043] 2. Enzyme digestion and recovery of DNA fragments
[0044] The buffer used for plasmid digestion is selected according to the Takara reagent catalog. For single digestion, use the buffer attached to the product, and for double digestion, select the most suitable buffer according to the reagent catalog. Generally, at least 1U enzyme is added to 1μg plasmid, and incubated at 37°C for 2h.
[0045] In this experiment, a kit was used to extract the plasmid, which has higher purity and better digestion effect. Generally, the digestion time can be cut in 1h, and it can be fully digested in 3-4h. It is better to digest enzymes without star activity overnight. ...
Embodiment 3
[0075] Example 3 Results and application of the expression of drought-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 digested with restriction enzymes XbaI and SmaI to connect the directional sticky end-blunt end, 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 and the control CK of the ground cover chrysanthemum variety "Wanfen" were planted in an incubator. Select 8-10 pieces of chrysanthemum seedlings at the true leaf stage for stress treatment, and control water for 4 days at 30°C and 30%RH. Chrysanthemum seedlings grown for 40 days were selected and treated with water control for 4 days, and their drought resi...
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