Poster: Transcription Regulation

Abs # 1049: Activation of 5' splice donor site on the leader sequence of peanut chlorotic streak virus (PClSV) by the antisense of PClSV ORF VII (p7R) leads to enhanced expression of genes in transgenic plants

Presenter:	Maiti, Indu B., imaiti@uky.edu
Authors	Maiti,  Indu B. (A)   Bhattacharyya, Somnath  (A)   Pattanaik, Sitakanta  (A)
Affiliations:	(A): University of Kenticky, KTRDC

The antisense orientation of the peanut chlorotic streak virus (PClSV) ORF VII (denoted as p7R), in conjunction with the sense orientation of the PClSV leader sequence, acts as an intron and enhanced the expression of a reporter gene, analyzed in protoplasts and transgenic plants. Correct 5' and 3' splicing sites were determined for intron removal from the chimeric constructs using either GUS or CAT as a reporter gene. Two identical consensus 5'-splicing donor sites (AG/GTATA) are located at positions (+147 to +153) and (+ 283 to + 289) from the transcription start site (TSS) of the PClSV full-length transcript (FLt). The p7R can activate only one donor site located at position (+ 283 to + 289) from the TSS on the leader sequence. The 3' splice site (TAG/GATT) is located on the p7R sequence at position (+785 to +791) from the TSS. The combination of PClSV FLt leader and p7R enhanced the expression of reporter genes (CAT and GUS) by as much as 30 to 800-fold in both protoplast transient expression experiments and stably transformed transgenic plants, compared to constructs containing sense orientation of PClSV ORF VII (p7). An increased level of mature transcripts accompanied this. This suggests that this combination of elements can mediate intron-mediated enhancement (IME) phenomenon. We also demonstrated comparative IME with other heterologous promoter from Caulimoviruses