Promoter studies of novel nanovirus like component associated with cotton leaf curl disease

A plant expression vector pCAMBIA 1301 was used. This vector has GUS gene with CaMV 35S promoter. GUS allows visualization of expression in particular cell type. Vector pCAMBIA 1301 was digested with KpuI and Bg/II restriction enzymes. A 670 bp fragment of CaMV 35S promoter and lac% alpha gene was separated on 1% agarose gel. The vector without 35S was cluted from agarose gel. PCR product was also digested with KpuI and Bg/II restriction enzymes. Digested PCR product and vector were used in ligation reaction. The ligation mix was transformed into E.coli DH5-alpha by electroporation. Recombinant plasmid was named as PGH-I. The plasmid pGH-I, (GUS with DNA-I promoter) and pCAMBIA 1301 (GUS with 35S promoter as a +ve control) were used in transient expression assay. Tobacco and cotton leaves explants were bombarded with these plasmids. DNA-I promoter was found to be active in both cotton and tobacco leaves explants as blue foci were found in transient expression assay. Comparatively high number of blue foci was found in tobacco leaves explants bombarded with DNA-1 promoter. Blue foci showing GUS expression found in mesophyll cells, guard ells epidermal cells and vein of leaves. GUS expression in other than phloem cells revealed that CLCuV DNA-1 promoter is not a phloem specific promoter. Promoter activity of DNA-1 was also checked in stem explants of tobacco and cotton. Blue foci showing GUS expression were found in stems explants of tobacco and cotton. These observations revealed that the DNA-1 promoter was active in both experimental and host plants. Cotton and tobacco are dicotyledonous therefore to check promoter activity in monocotyledonous plant rice embryos were bombarded with pGH-I and pCAMBIA 1301. High number of blue foci was found in rice embryos bombarded with pGH-I as compared to pCAMBIA 1301. This observation proved the strong activity of DNA-1 promoter in rice embryos. Rice and cotton calli were also used in transient expression assay. High number of blue stained foci showing GUS expression was observed using either promoter. In cotton calli few blue stained foci were observed, this perhaps because of low transformation efficiency in cotton. The promoter activity of DNA-1 was also assayed using Agrobacterium mediated transient expression. The plasmid pGH-I and pCAMBIA 1301 were transformed into Agrobacterium tumefaciens strain LBA 4404. Tobacco leaves were infected with Agrobacterium by agroinfilteration. Infected leaves showed intensely blue stained using either promoter. The activity of DNA-1 promoter was comparable with CaMV 35S promoter. Similar GUS expression of DNA-I promoter with respect to CaMV 35S promoter revealed that CLCuV DNA-1 promoter may have a strong promoter activity and constitutive in nature. The DNA-I promoter was found to he active in both monocotyledonous and dicotyledonous crop plants.