Caulimo viruses as vector
- 2. Plant Viruses: • Plant viruses are considered as efficient gene transfer agents as they infect the intact plants and amplify the transferred genes through viral genome replication • They are non integrative vectors Eg, Pepper mint mottle virus, Leaf curl virus Cauliflower Mosaic Virus (Caulimovirus) • Cauliflower mosaic virus (CamV) belongs to the group caulimovirus, can be used as potential candidate to deliver foreign gene into the plant. • It is perhaps the best studied viruses among plant virus, which infects several members belonging to Cruciferae family. • As an infective agent, can cause disease in wide range of commercially important cultivated crops. • Cauliflower mosaic DNA has been subjected to a wide range of manipulation. • This was the only and first virus to be manipulated and used as a favourable choice for genetic engineering work.
- 3. CaulimoViruses • Caulimoviruses contain circular ds DNA and are spherical in shape • This group includes 15 viruses of which CaMV is the most important for gene transfer • The other caulimoviruses includes carnation etched virus, dahlia mosaic virus mirabilis mosaic virus and strawberry vein banding virus • CaMV is a plant virus that infects mostly brassicaceae family( cauliflower and turnips) and Solanaceae species • CaMV is transmitted in a non circulatory manner by aphid species Mysus • It is a icosahedron structure with a diameter of 52nm built from 420 cap protein subunits
- 4. Genome of CaMV • It has circular ds DNA containing 8kbp • The promoter 35S RNA is a very strong constitutive promoter responsible for the transcription of the whole CaMV genome. • It is well known for its use in plant transformation. • It causes high levels of gene expression in dicot plants • The 35S RNA is particularly complex, containing a highly structured 600 nucleotide long leader sequence . • This leader is followed by seven tightly arranged, longer ORFs that encode all the viral proteins. • The mechanism of expression of these proteins is unique, in that the ORF VI protein (encoded by the 19S RNA) controls translation reinitiation of major open reading frames on the polycistronic 35S RNA, a process that normally only happens on bacterial mRNAs
- 5. Criteria needed for vector: • The virus must be capable of spreading from cell to cell through plasmodesmata • The viral genome should be able to replicate in the absence of viral coat protein and spreads from cell to cell •Elicit little or no disease symptoms • Should have broad range of host
- 6. CamV Vector: • Cauliflower mosaic virus can be used as a potential vector due to the infective nature of its genetic material. • This could be proved by applying viruses on the leaf rubbed with abrasive material. The CamV cannot accommodate foreign DNA, if the size exceeds its normal size. • The inserted DNA may destabilize infectious nature of the virus. • Other constraints are the packaging of genome and limitation of the insertion of foreign DNA. • Despite the marginal constraints, CamV genome can be packed in nucleosome and is able to undergo transcription by plant RNA polymerase II. • For effective transmission of CaMV the foreign DNA must be encapsulated in viral protein and the inserted gene should not interfere with native assembly of virus • CaMV does not contain any non coding region wherein foreign DNA can be inserted
- 7. • The genome of CamV consists of six major and two minor open reading frames (ORF), in tightly packed arrangements. • The two ORF regions, one (ORFII) codes for insect transmission factor and other (ORF VII) with unknown functions can be replaced with gene of interest. • So the gene of interest can be replaced in the place of these two genes • Use of CaMV as a vector pertains to the bacterial dhfr (dihydroxy folate reductase) gene inserted in the place of gene II and successfully expressed in plants • This dhfr gene is needed for providing resistance to methotrexate( inhibitor of dhfr and extremely toxic to plants) Limitation of CaMV: • Limited capacity for insertion • Infective capacity is lost if few hundreds of nucleotides are introduced • Because of its exceeding natural genome size they are not effectively suitable for gene transfer