Development of Efficient Synthetic Promoters derived from Plant Pararetroviruses

Abstract

Promoters are specific sequence of nucleotides present upstream of the gene coding region; comprises of multiple cis-acting elements having specific binding for transcription factors which are involved in initiation and regulation of gene expression. The basic strength of promoter lies in the notation and arrangement of these cis-acting elements and their combinational interaction with transcription factor. Therefore, strong promoters could be developed by redesigning their architecture, which may be exploited for strong expression of ectopic gene(s). In the present study a set of eight chimeric promoters were synthesized as g-block gene fragments employing two well-established approaches, domain swapping and hybridization. Important domains of previously characterized Rice Tungro Bacilliform Virus (RTBV) and Mirabilis Mosaic Virus (MMV) truncated promoter were exploited for the development of novel synthetic promoters. The promoter constructs were cloned upstream of a GUS and GFP reporter gene to evaluate the efficacy in two independent system i.e. monocot and dicot plant in addition to bacterial system. Gus assay of electroporated tobacco protoplast (Xanthi-Brad) and agro-infiltrated tobacco, petunia, rice and millet was performed for transient expression studies. In protoplast expression analysis 3.56 and 2.5 times higher activity than CaMV35S were obtained in pUPMA-RP1-MP1GUS and pUPMA-RP4-MP1GUS respectively. In comparison of CaMV35S promoter, 0.87 and 0.68 fold increase in expression were observed in RP1-MP1 and RP4-MP1 respectively in transgenic tobacco while 0.95 increase was found in RP1-MP1 transgenic rice. On contrary pUPMA-RP1-MP1GFP construct had shown highest expression in plants whereas in bacterial system pUPMA-RP4-MP1GFP promoter construct had shown strongest expression. newlineBiobeads having encapsulated bacterial cells, expressing HMP3 gene in regulation of pUPMA-RP4-MP1 promoter was found to reduce 72.9% and 29.2 % Copper (Cu) and Zinc (Zn) concentration from waste water. From the obtained results it might be concluded that developed synthetic promoter would be efficiently used for translational research in both monocot/dicot plants and bacterial system for ectopic gene expression. newline newline