Cloning and transcriptional activity analysis of U6 promoter in sweetpotato
In the CRISPR/Cas9 gene editing system,the U6 promoter can edit target genes by driving sgRNA expres-sion,so its transcriptional activity can affect the efficiency of gene editing.Although studies on cloning and application of U6 promoter have been carried out in Arabidopsis thaliana,corn,soybean,cotton and other plants,there aren't any reports on relative research of U6 promoter in sweetpotato.In this study,we searched the Ipomoea trifida genome database for candidate U6 RNA,using the conservative sequence of AtU6 SnRNA in A.thaliana,and then found two different candidate U6 promot-ers with lengths of 526 bp(IbU6p-1)and 532 bp(IbU6p-2),respectively.Through sequence alignment analysis,we found that the U6 promoter of sweetpotato has upstream sequence element(USE)and TATA box,and its sequence was highly simi-lar to that of A.thaliana.Then,a recombinant box U6::LUC was constructed using the obtained U6 promoter nucleic acid sequence to drive the expression of firefly luciferase gene.Finally,Agrobacterium tumefaciens containing the recombinant vec-tor was genetically transformed into Nicotiana benthamiana leaves and sweetpotato callus tissues by transient expression,and luciferase activity was analyzed using fluo-rescence imaging technology.The results showed that both sweetpotato U6 promoters could drive the expression of LUC gene and had transcriptional activity in tobacco and sweet-potato calli.At the same time,the transcriptional activities of IbU6p-2 in tobacco leaves and sweetpotato calli were signifi-cantly higher than that of A.thaliana U6 promoter.The results of this study lay a foundation for further development of sweet-potato gene editing technology.
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