Application of AtTGA 4gene in improving plant adverse resistance
A plant and gene technology, applied in the application field of AtTGA4 gene in improving plant stress resistance, can solve problems such as the unresearched AtTGA4 transcription factor, and achieve the effect of improving nitrogen absorption capacity
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Embodiment 1
[0078] Example 1, Changes in the expression level of AtTGA4 gene under drought and low nitrogen conditions
[0079] 1. Expression analysis of AtTGA4 gene under drought conditions
[0080] 1. Extraction of Arabidopsis total RNA
[0081] The seeds of wild type Arabidopsis Columbia0 were treated with 70% (volume fraction) alcohol in an ultra-clean workbench for 3 minutes, and then washed twice with sterile water, each time for 1 minute, with 5‰ (mass volume ratio) sodium hypochlorite. After sterilizing for 10 minutes, wash twice with sterile water for one minute each time. Sow sterile seeds on MS with a toothpick 0 On a solid medium plate, after vernalization at 4°C for 3 days, transfer to a tissue culture room to grow for 1 week, select seedlings of the same size and transfer to MS containing 8% PEG (mass volume ratio) 0 Treatments were initiated on solid medium plates. The processing time was set to 0h, 0.25h, 0.5h, 1h, 3h, 6h, 12h, 24h, respectively.
[0082] The treated ...
Embodiment 2
[0097] Embodiment 2, the cloning of AtTGA4 gene and the acquisition of overexpression strain
[0098] 1. Cloning of AtTGA4 gene and construction of recombinant expression vector pBI121-TGA4
[0099] The wild-type Arabidopsis Columbia0 at the adult plant stage was used as the experimental material, and the total RNA was extracted by the TRNzol method, and the qualified RNA was reverse-transcribed into cDNA with the rice transcription kit of TaKaRa Company, and stored at -20°C for future use.
[0100] Specific primers TGA4-F2 and TGA4-R2 were designed according to the CDS sequence of the AtTGA4 gene provided on the TAIR website to amplify the full length of the AtTGA4 gene. The reaction system includes 2×GC Buffer 25μl, dNTP Mix 4μl, upstream and downstream primers 1μl, primerstar 0.3μl, ddH 2 O 16.7 μl, template cDNA 2 μl (the above PCR reaction reagents are from TaKaRa company PCR amplification kit). The PCR amplification program was pre-denaturation at 94°C for 10 min, dena...
Embodiment 3
[0109] Embodiment 3, the drought resistance identification of transgenic Arabidopsis thaliana
[0110] 1. Detection of drought resistance in germination stage
[0111] The following three Arabidopsis plants were used as experimental materials: wild-type Arabidopsis Columbia0 (WT), four T 2 Transgenic AtTGA4 gene strains (TGA4-1, TGA4-2, TGA4-3, and TGA4-4), and Arabidopsis thaliana transformed with pBI121 empty vector, after aseptic treatment, they were sown on MS 0 Solid medium and MS containing 8% (w / v) PEG 0 On solid medium, after vernalization at 4°C for 3 days, put them in a tissue culture room to grow. When the treatment reached 10 days, the number of seedlings with 2 green leaves was counted. When the treatment continued to 17 days, the differences in phenotype were observed and root scans, stem length and Leaf area measurement. The experiment was repeated 3 times, and the results were averaged.
[0112] The results showed that after 10 days of treatment, the number...
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