Molecular interaction and gene expression pattern of CPSF subunits and differential display analysis under fungal induction in tomato

Abstract

Gene expression in a cell is mainly controlled by regulating transcription of mRNA. 3’- end polyadenyation is a key step in mRNA biogenesis. It involves interaction of poly(A) factors with the poly(A) sites and with each other. Understanding such interactions will have vital implications in regulating gene expression levels. Tomato, Solanum lycopersicum, is an important member of the Solanaceae family and grown worldwide with high production. However, regulation of gene expression, especially at polyadenylation level has not been studied in tomato. Cleavage and Polyadenylation Specificity Factor (CPSF) has a central role in cleavage and polyadenylation process. The present proposal therefore was planned for the study of CPSF subunits and genes in tomato using various strategies. CPSF gene expression pattern was studied from tomato seeds, leaflets and roots by isolating RNA followed by RT-PCR. The expression level of these genes was studied on automated gel electrophoresis system (Experion). Highest expression was found for the subunit CPSF 73II followed by CPSF73I. The gene expression levels of the CPSF subunits were highest in the seedlings as compared to those in leaflets and roots. Effect of fungal stress on CPSF and differential gene expression were also studied with the help of gene specific and arbitrary primers respectively. Time course under fungal stress showed highest expression of CPSF 73II followed by CPSF73I gene after 4 and 6 hours post inoculation respectively. Fungal

stress reduced the expression of all these genes. Differential gene expression under fungal stress showed expression of many useful genes. Molecular interactions among CPSF subunits were examined by proteomics approach following pull-down of the protein complex. The CPSF subunit cDNAs were transformed in Agrobacterium followed by transformation in tomato suspension culture. Proteomics results have shown the interactions among CPSF subunits with each other and with other neighboring proteins. Outcome of the project is expected to have important implications for the control of gene expression in tomato to improve yields and varieties.