Photoinduction promoter for promoting specific expression of alfalfa plastocyanin and primers for quickly cloning promoter
A light-induced promoter and plastocyanin technology, applied in the field of medicine, can solve the problems of plant variation and affecting the normal growth and development of plants
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[0019] (1) PCR method to clone the promoter sequence;
[0020] a) Reaction system;
[0021] Sterilized deionized water 33.75ul, alfalfa genomic DNA 5ul, 10×PCR buffer 5ul, dNTPmix 4ul, upstream and downstream primers 1ul each, rTaq 0.25ul;
[0022] b) Reaction conditions: pre-denaturation at 95°C for 2min, denaturation at 95°C for 30s, annealing at 53°C for 30s, extension at 72°C for 1min, cycle 30 times, extension at 72°C for 10min;
[0023] c) Two-way primer upstream primer 5'-CCCAAGCTTAAAATAATAATGTGTGTTATGAGA-3', downstream primer 5'-cgggatccgctctgacacaaagcctt-3'.
[0024] (2) PCR reaction products were detected by agarose gel electrophoresis and sent to Gene Company for sequencing and sequence analysis;
[0025] (3) Construction of recombinant plasmids for transformation; including a) extraction of plasmids; b) enzyme digestion; c) enzyme linkage; the characteristics are: a) SDS alkali denaturation method to extract p BI121 plasmid; b) by Hind Ⅲ-BamHI double enzyme dige...
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