Lecture 7.ppt

Lecture 7 Manipulation of foreign gene and secretion of foreign protein Manipulation of gene expression Isolation and Use of Different Promoters Targeted Alterations in Plant DNA Targeting foreign DNA into chloroplast genome Isolation and Use of Different Promoters Use of specialized vectors, called promoter tagging labeling vectors approach relies on the Agro bacterium mediated Ti plasmid transformation system a promoter less reporter gene is placed next to the right border of the Ti plasmid vector after transfer of the T-DNA into a plant chromosome the reporter gene from the vector is situated adjacent to the plant DNA if the T-DNA is inserted at the promoter region of a functional gene, transcription of the reporter gene occurs Isolation and Use of Different Promoters For eg: neomycin phosphotransferase npt gene as a reporter expression detected by selecting kanamycin-resistant transformants Fig A difficult to identify tag a promoter that is active only during a certain developmental stage or that is induced by a specific environmental factor Isolation and Use of Different Promoters To overcome this, two-gene selectable marker system was devised Fig B In this case, a hygromycin resistance gene was placed under the control of a constitutive promoter next to a promoter less reporter gene within the T-DNA After hygromycin-resistant transformants are selected, the transformants can be checked by an enzyme assay under different conditions for expression of the reporter gene Targeted Alterations in Plant DNA use of chimeric oligonucleotides delivery into a plant cell by microprojectile bombardment DNA repair enzymes recognize the mismatches between the targeted gene and the chimeric oligonucleotide During the repair process, the altered DNA is incorporated into the plant genome detected phenotypically similar to conventional mutagenesis and selection procedures An example of a chimeric oligonucleotide used to change the nucleotide sequenc