Cloning And Transcriptional Activity Analysis Of U6 Promoters In Apple

CRISPR/Cas9 genome editing technology has been successfully applied to a variety of plants and has a broad application prospect in plant molecular breeding.At present,the application of gene editing technology has made slow progress in apple?Malus×domestica?.U6 promoter is an important element for the transcription of sgRNA in the CRISPR/Cas9 genome editing system,and its transcriptional activity may be affected by genetic relationship and sequence length.Therefore,selecting an apple U6promoter with high transcriptional activity and suitable fragment size would provide a basis for the optimization of apple CRISPR/Cas9 gene editing technology system.There are multiple U6 snRNAs in the‘Golden Delicious'genome.The six U6 snRNAs with with E-value less than 3e^-40 in the apple genome were selected in this study,and 1 500+27 bp U6 promoters containing 1 500 bp upstream of the transcription start site and 27 bp additional coding region were selected as candidate promoters to perform sequence alignment.Then,the candidate U6 promoters were cloned and constructed them into an expression vector for the firefly luciferase?LUC?gene.Then,the apple calli and tobacco?Nicotiana benthamiana?leaves were transformed by Agrobacterium-mediated transient transformation.The transcriptional activities of U6 promoters were determined according to the luciferase activity.After selecting a U6 promoter with the highest transcriptional activity,it was truncated with different lengths of 5'ends,and its transcriptional activity was analyzed by the same method mentioned above.The main results were as follows:1.Six U6 snRNAs with E-value less than 3e^-40,which were located on chr 6?chr 7?chr 9?chr 10?chr 15 and chr 17 respectively,were selected from the‘Golden delicious'genome for further experiments.Sequence comparation among six candidate 1 527 bp U6 promoters and the Arabidopsis U6-1 promoter was conducted,results showed that all apple U6 promoters contained the conserved-60USE motif and-30 TATA-like Box and their relative positions were also very conservative,and it also had been found that the transcription start site of all U6 promoters are‘G',the same as Arabidopsis.2.Luciferase activities were detected in tobacco leaves and apple calli after transient transformation,and transcriptional activities of 6 candidate apple U6 promoters were compared by fluorescence signal acquisition.The results showed that all six candidate apple U6 promoters detected fluorescent signals,of which U6 promoter had the strongest fluorescent signal on chromosome 10.Then,the same method was used to compare the transcriptional activity of the U6 promoters which were shorted at 5'end?1500,959,275 and 116 bp in length?on chromosome 10.The result showed that the length of 275 bp U6promoter had the highest transcriptional activity.3.In apple calli,comparing the transcriptional activity of the selected apple U6 promoter with the Arabidopsis U6-1 promoter,it was found that,the transcriptional activity of apple U6 promoter in apple calli was significantly higher than that of the Arabidopsis U6-1 promoter.In summary,a short sequence length and high transcriptional activity of apple U6 promoter was selected from the apple genome in this study,which can provide candidate U6 promoter for optimization of apple CRISPR/Cas9 gene editing system.