Cloning and Activity Analysis of U6 Promoter in Pineapple
Single guide RNA(sgRNA)is the"Command center"of CRISPR/Cas9 system,which can guide Cas9 to edit DNA at a fixed point.Its transcription ability directly affects the efficiency of gene editing.U6 promoters are often used to drive the expression of sgRNA,especially the endogenous U6 promoters of own species usually have more efficient starting efficiency.In order to screen out the endogenous high-efficient transcriptional activity U6 promoter of pineapple and improve the gene editing efficiency of pineapple CRISPR/Cas9,five endogenous U6 promoters were cloned from the pineapple genome,three U6 promoters were selected from rice,and a fusion ex-pression vector driving firefly luciferase(LUC)gene was constructed,the tobacco leaves was transiently trans-formed,and the transcriptional activity of each promoter was compared by detecting the biochemical luminescence activity.The results showed that five U6 promoters were cloned from pineapple genome and named AcU6-lP,AcU6-2P,AcU6-3P,AcU6-4P and AcU6-5P respectively.Biochemical luminescence signal detection found that five AcU6 had transcriptional activity,of which the AcU6-2P promoter drives the strongest fluorescence signal of LUC expression,followed by AcU6-5P promoter.Biochemical luminescence activity analysis showed that the ac-tivity of luciferase driven by AcU6-2P promoter was the highest,and AcU6-5P was second only to AcU6-2P,which was consistent with the fluorescence signal.To sum up,two endogenous U6 promoters AcU6-2P and AcU6-5P with high transcriptional activity were screened from the pineapple genome,which provided a basis for the optimization of the subsequent pineapple CRISPR/Cas9 gene editing system and molecular breeding.
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