Borage promoter and application thereof
A technology of promoter and borage, applied in the field of genetic engineering
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Embodiment 1
[0019] Example 1 Promoter and cloning and element analysis
[0020] Healthy borage leaves were cut, ground in liquid nitrogen, and genomic DNA was extracted by CTAB method. A pair of primers for PCR amplification were designed and synthesized according to the sequence of the known Δ-6 fatty acid dehydrogenase gene promoter:
[0021] Forward primer: 5'-AACCGTCAATAATGATGAAAATTTTG-3';
[0022] Reverse primer: 5'-TGAATAAAATATAAGAGGGTAGACTTTAAG-3'.
[0023] The PCR reaction system was as follows: 4.0 μL of cDNA, 5.0 μL of 10×Pyrobest Buffer, 2 μL of 2 mmol / L dNTP, 0.5 μL of Pyrobest enzyme, 2.0 μL of 10 μmol upstream and downstream primers, and the final volume was 50 μL.
[0024] The PCR reaction program was: pre-denaturation at 94 °C for 7 min, followed by 30 cycles: 94 °C for 30 s, 55 °C for 30 s, 72 °C for 90 s, after the cycle was completed, an extension reaction at 72 °C for 10 min, followed by incubation at 4 °C.
[0025] 1% agarose gel electrophoresis to detect PCR produ...
Embodiment 2
[0030] Example 2 Regulation of genes by promoters
[0031] 1. Vector Construction
[0032] Synthetic promoter-GUS gene, with an EcoRI restriction site added upstream and a Bg1II restriction site added downstream. The above gene was transformed into the pCAMBIA-1303 vector which also removed the 35S promoter by double enzyme digestion to obtain the pCAMBIA1303-proBoD6D vector. Subsequently, the pCAMBIA1303-proBoD6D positive vector was transfected into Agrobacterium tumefaciens EHA105. The following sequences are used as primers to detect positive transformants:
[0033] proBoD6D-FP (EcoRI):
[0034] 5'-GAATTCAACCGTCAATAATGATGAAAATTTTG-3';
[0035] proBoD6D-RP (Bg1II):
[0036] 5'-AGATCTTGAATAAAATATAAGAGGGTAGACTTTAAG-3'.
[0037] The PCR reaction system was as follows: 0.2 μL of plasmid, 5 μL of 10×Pyrobest Buffer, 2 μL of 2 mmol / LdNTP, 0.5 μL of Pyrobest enzyme, 2.0 μL of 10 μmol upstream and downstream primers, and the final volume was 50 μL.
[0038] The PCR reaction p...
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