Name                                               : Hatem Mohamed El-Mahdy    

Date of birth                           : 10/9/1978 AC.

Nationality                              : Egyptian.

Previous Degrees                  : Demonstrator.

Registration Date                  : 2005.

Awarding Date                       : 2008.

Supervisors                            : - Prof. Dr. Hala M.Y. Habib.

                                                  - Prof. Dr. Taymor M. Nasr El-Din

Examiners                              : - Prof. Dr. Panayota A. Kyriako Poulou

                                                  - Prof. Dr. Ali M.M. Abd El-Salam

Title of Thesis                        : BIOLOGICAL AND MOLECULAR STUDIES ON A

  LOCAL ISOLATE OF POTATO VIRUS Y.

Keywords: PVY, Detection, Polymerase chain reaction, ELISA, Inclusion bodies,

        rep gene, sequence

Summary:

We are reporting here some serological, biological and molecular tools for detection and identification of a PVY isolate. On the side of serological studies a number of 109 infected potato samples exhibited different virus like symptoms were collected from three different locations from open fields near Kafr El-Zayiat city, Egypt and tested by indirect DAS-ELISA. Results showed that 53, 5 and 28 out of 100 infected potato plants showing +ve reactions with PAbs specific to PLRV, PVX and PVY antisera, While 9 samples showed –ve reactions. In case of biological studies, the viral isolate under investigation gave faint mosaic on Solanum tuberosum L. leaves, mosaic on Datura metel leaves, necrotic local lesions on Chenopodium quinoa and Veinal necrosis on Nicotiana tabacum L. cv. (White Burely) leaves. Also, the physical properties showed that the viral isolate under investigation has TIP = 60^oC, DEP = 1:2 x 10⁴ and withstand storage at 0^oC up to 6 months while completely inactivated after 50 h. for it’s resistance to aging. Flexuous rod-shaped virus like particles were found when the purified virus preparation was stained with 2% uranyl acetate and subjected to electron microscopy. Light and electron microscopy showed that PVY of this study induced inclusion bodies that appeared as: pinwheels and cylindrical inclusions of laminated aggregates in the cytoplasm of virus infected cells. Also, some cytopathological effects on the virus infected cells could be summarized in effects on chloroplasts and mitochondria. On the level of molecular studies, the total RNA was isolated from PVY infected Solanum tuberosum L. leaves and then used as a template for RT-PCR to amplify the cDNA followed by amplification of rep gene (1.5 Kbp). After extraction & purification of the PCR product, it was cloned into the plasmid. This plasmid was then introduced into E. coli strain JM109. PVY rep gene that inserted into purified DNA plasmid (pRT₁₀₄ which named pREP) was confirmed by PCR and restriction endonuclease analysis. This followed by sequencing of PVY rep gene and comparing it with (23) PVY overseas strains or isolates. The similarity between our PVY Egyptian isolate and the (23) overseas strains or isolates ranged from 83 to 99% and from 89 to 97.9% based on the level of DNA and deduced amino acids sequence, respectively. In conclusion, our Egyptian virus isolate is PVY strain N.

 

 

Rights of Design © is reserved  to Faculty of Science

Designed and Directed by  A.H.A