Functional characterization of a strong bi-directional constitutive plant promoter isolated from cotton leaf curl Burewala virus

• Published in: PloS one 2015-03, Vol.10 (3), p.e0121656-e0121656
• Main Authors: Khan, Zainul A, Abdin, Malik Z, Khan, Jawaid A
• Format: Article
• Language: English
• Subjects: Arabidopsis; Base Sequence; Begomovirus - genetics; Chemical properties; Coat protein; Confocal; Confocal microscopy; Cotton; Crop diseases; Deoxyribonucleic acid; DNA; Ectopic Gene Expression; Fluorescence; Gene expression; Genes; Genetic Engineering - methods; Genomes; Genomics; Gossypium - genetics; Gossypium hirsutum; Green fluorescent protein; Green Fluorescent Proteins - genetics; Laboratories; Leaves; Microscopy; Molecular Sequence Data; Nicotiana - genetics; Plant cells; Plant Leaves - genetics; Plants - genetics; Promoter Regions, Genetic - genetics; Promoters; Proteins; Scanning microscopy; Sequence Analysis; Single-stranded DNA; Tobacco; Transcription factors; Transcription, Genetic; Transformation, Genetic; Transgenes; Transgenic plants; Viral Proteins - genetics; Virology; Viruses
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0121656

Cotton leaf curl Burewala virus (CLCuBuV), belonging to the genus Begomovirus, possesses single-stranded monopartite DNA genome. The bidirectional promoters representing Rep and coat protein (CP) genes of CLCuBuV were characterized and their efficacy was assayed. Rep and CP promoters of CLCuBuV and 35S promoter of Cauliflower mosaic virus (CaMV) were fused with β-glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes. GUS activity in individual plant cells driven by Rep, CP and 35S promoters was estimated using real-time PCR and fluorometric GUS assay. Histochemical staining of GUS in transformed tobacco (Nicotiana tabacum cv. Xanthi) leaves showed highest expression driven by Rep promoter followed by 35S promoter and CP promoter. The expression level of GUS driven by Rep promoter in transformed tobacco plants was shown to be two to four-fold higher than that of 35S promoter, while the expression by CP promoter was slightly lower. Further, the expression of GFP was monitored in agroinfiltrated leaves of N. benthamiana, N. tabacum and cotton (Gossypium hirsutum) plants using confocal laser scanning microscopy. Rep promoter showed strong consistent transient expression in tobacco and cotton leaves as compared to 35S promoter. The strong constitutive CLCuBuV Rep promoter developed in this study could be very useful for high level expression of transgenes in a wide variety of plant cells.