Application of trifoliate orange protein kinase PtrSnRK2.4 in plant drought-resistant genetic improvement
A plant and protein technology, applied in the field of plant genetic engineering, to reduce agricultural production costs and achieve environmental friendliness
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Embodiment 1
[0026] Example 1: Cloning of full-length cDNA of PtrSnRK2.4 gene of Citrus trifoliate
[0027] The cDNA of Hovenia trifoliate was used as a template to amplify with high-fidelity enzymes. The sequences of the amplification primers were as follows: forward primer: 5’-TGGATTGAGATGGAAGAGAGA-3’, reverse primer: 5’-CGGATCATGGCTCATAATATC-3’.
[0028] AxyPrep-96 DNA Gel Recovery Kit (Axygene, USA) was used to purify and recover the amplified product, and then the purified product was connected to the pEASY-Blunt vector (full type gold, China). See the connection system and incubate at room temperature for 5 minutes before transformation Escherichia coli competent DH5α. Spread the plate, culture it upside down at 37°C, pick and shake the bacteria, and carry out positive detection. After obtaining the positive clone, send it to Wuhan Qingke Biological Co., Ltd. for sequencing. According to the sequencing result, it is the full-length sequence of the PtrSnRK2.4 gene.
[0029] Sequencin...
Embodiment 2
[0030] Example 2: Analysis of the expression of PtrSnRK2.4 gene under different conditions
[0031] The 2-month-old citrus aurantium seedlings were planted in a constant temperature and sunlight incubator. Select seedlings with consistent growth, uproot them, wash them with clean water, insert them into a triangular flask filled with deionized water, and place them in an incubator with a temperature of 25°C and an alternating photoperiod of 16h / 8h for three days. For the dehydration treatment, the water-cultivated seedlings were wiped off the residual water droplets with filter paper, and placed on a 90×90mm filter paper for dehydration for 0h, 3h, 6h, 9h, 12h, 24h, and 36h. During ABA treatment, the water-cultured seedlings were placed in 100 μM ABA solution, and the treatment sampling time was 0h, 3h, 6h, 9h, 12h, 24h, and 36h, respectively. In the case of dehydration treatment+FLU (ABA inhibitor) treatment, the hydroponic seedlings were pre-cultured in 100 μM FLU solution ...
Embodiment 3
[0034] Example 3: Subcellular localization of PtrSnRK2.4 gene
[0035] Amplify the ORF region of PtrSnRK2.4 (without stop codon), design amplification primers (F: 5'-GGACTAGTTGG ATT GAG ATGGAAGAGAGAT-3' and R: 5'-ACGCGTCGAC CGGATCATGGCTCATA TA-3') and fuse to the vector On p101LYFP, the YFP protein is located at the C-terminus of the gene, and its expression is driven by the CaMV35S promoter. Then, the control 35S:YFP+35S:OFP-HDEL and 35S:PtrSnRK2.4-YFP+35S:OFP-HDEL were transiently transformed into the leaf epidermal cells of Nicotiana benthamiana respectively, and the fluorescence of the control was observed by confocal laser microscopy, and it was found that the fluorescence of the control filled the whole area. Epidermal cells, including the cell membrane and nucleus, compared with this, the yellow fluorescence can also be observed in the cell membrane and nucleus after the fusion expression vector 35S:PtrSnRK2.4-YFP is transferred into this type of tobacco, and the fluore...
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