Annual Report 2011-2012 - Nabi.res.in

NATIONAL AGRI-FOOD BIOTECHNOLOGY INSTITUTEFigure 15: Gene expression analysis in wheat leaves after 21 dpi with WDIV. (A) Up and down regulation of genes. (B)Venn diagram representing the set of genes up regulated and down regulated during virus infection. (C) Hierarchicalclustering and changes in gene expression in virus only or virus+satellites-infected wheat plants at 21st day post inoculation(dpi). Gene tree maps were generated using k-mean clustering at 2 fold or higher differentially regulated wheat genes.6. Development of VIGS vector by accession number FM998042). The PDSmodifying the viral genome: Two fragment was excised from the cloningmodifications were done in viral genome by vector and was inserted into the MCS ofremoving a small stretch of nucleotides and viral genome using SpeI restrictioninserting Multiple Cloning Sites (MCS) at endonuclease. The viral genome havingthe same positions. The modified viral PDS was termed as VIGS-PDS. In order togenome was cloned in a plasmid vector for infect wheat for silencing of the PDS gene,further manipulation.head-to-tail dimerization of VIGS-PDS wasdone into pCAMBIA1301 binary vector.7. Validation of VIGS vector using visualThe PDS silencing cassette (pCAMBIAmarkers:A 327 bp fragment of phytoeneVIGS-PDS) was transformed intodesaturase (PDS) gene was amplified usingAgrobacterium tumefaciens (straincDNA as a template prepared from wheatGV3101). The empty VIGS vector was also(Triticum aestivum L.). The amplifiedtransformed in Agrobacterium. Thefragment was cloned and sequenced tosilencing cassette and empty VIGS-vectorestablish the identity. Sequence analysiswere inoculated to healthy wheat plantsshowed an identity of 99% with theseparately and scored for phenotype at 15previously reported PDS gene (GenBankdays (Figure 16 A, B and C).TAATATT/AC V E CV2IntronC2C1WDIV2783 bpV1Figure 16: Modification of WDIV for functional tool. (A) Genome organization of WDIV detected fromwheat in India and used for developing as VIGS-vector, C1-Rep Protein, C2-Rep-A Protein, V1-Coat Protein,V2-Movement Protein. (B and C) Leaves from silencing PDS-cassette inoculated (V), empty VIGS-vector(E) and uninoculated control (C) wheat plants. (C) Partial view of a VIGS-PDS inoculated wheat plant.15