Expression studies of herbal proteins to suppress PVY in Potato

Abstract

This dissertation presents the expression studies of herbal proteins to suppress PVY in Potato. The experimental investigations start with the isolation of coat protein (CP) gene of potato virus Y from the PVY infected potato plants. The CP gene has been cloned in mammalian expression vector pcDNA 3.1 (+) and transfected in mammalian cell line (HepG2). Five herbal proteins were isolated and purified through ion exchange chromatography. The effect of purified herbal proteins on the down regulation of messenger RNA of CP gene in transient assay has been investigated. Four of the purified herbal proteins has down regulating effect on messenger RNA of CP gene up to 99%. Furthermore, the in-silico protein-protein interaction of CP protein with Crocus vernus and Brassica rapa have been studied. The genes of Crocus vernus lectin protein and Brassica rapa lectin protein have been cloned in pCAMBIA 1301 plant expression vector to study the transformation of CVL and BRL genes in potato. The pCAMBIA 1301 with CVL and BRL genes were then transformed in Agrobacterium tumefaciens through freeze thaw method. Colonies were picked individually and harvested in YEP media containing Kanamycin as an antibiotic in incubator shaker at 28˚C for 24 hours. Potato plants of age of 3 to 4 weeks grown in the culture tubes, in vitro media have been used for the transformation purpose. Plant upper nodes of size 2-3 mm in length as explants used for the transformation. The explants were arranged and grown on Murashige and Skoog medium (MS-zero media) in plates before the process of transformation. The temperature of the growth room was maintained ~24˚C with the help of light intensity of 2000-3000 Lux for a period of 16 hours. Co-cultivation and regeneration of Explants with Agrobacterium have been investigated. GUS Assay of Transgenic Plants, DNA Dot Blot Hybridization Assay VIII and PCR amplification studies have been carried out to confirm the transformation of cloned genes. Artificial inoculation of PVY on transgenic along with control plants has been performed and confirmed via ELISA Assay after thirty days of post inoculation. Real time PCR studies were conducted to investigate the effect of antiviral genes against PVY in transgenic potato plants versus control plants. Real time PCR studies further confirm the downregulation of the CP gene in the TPb-04 line up to 40%. In vitro assay, CP gene downregulation by the CVL and BRL was 75-99%. The observed change in transgenic cassette and expression level might be due to the insertion effects of the CVL and BRL in the potato genome or due to heavy artificial infection of the PVY of the transgenic plants as compared to natural inoculums which is mostly low and spread gradually through the vector.