Lecture 9 - PLB 113 Lecture 9 III Gene Transfer and...

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PLB 113 – Lecture 9 III. Gene Transfer and Epigenetic C. Development of Gene Transfer System D. Transgene Expression E. Cosuppression and Epigenetics Clicker #1 : If you transfer a bacterial gene and its promoter into a plant, will it be expressed? No Promoter of bacterial gene is not recognized by the plant cell. Thus, bacterial gene will not be expressed. Agrobacteria has adapted the genes (TMS, TMR) in T-DNA. These genes are present in the bacteria but not expressed. The reason why they are present in the bacteria is when they are transferred to plant cell, they can be expressed in the plant cell and takes over the plant cell = genetic colonization. ----------------------------------------------------------------------------------------------------------------------------- - Desired characteristic of transfer system #2 : Selection of transformed cells - this step is required since the efficiency of transferring gene is not 100% (it’s only 20% to 0.0001%) - people take advantage of: 1. herbicide resistance gene 2. antibiotic resistance gene * antibiotic = class of molecules that kill prokaryote * antibiotic gene comes from bacteria * ex. Neomycin phosphotransferase (NPT II), which is the antibiotic resistance gene that inactivate Kanamycin (antibiotic that inhibits bacterial protein) Transferring the bacterial gene with its promoter into a plant directly will not result the expression of this particular gene in the plant cell. In order to have this antibiotic-resistance gene expressed in the plant => Chimeric gene. Idea of Chimeric Gene: 1. Take the promoter + 5’ UTR and 3’UTR + 3’ flanking region has the signal for where the poly-A will be added) * 5' UTR => contains sequence important for START of mRNA * 3' UTR => sequence signaling where poly-A added to mRNA * 3' flanking region => indicates where transcription should be determined 2. Combine the regions of NOS plant gene (eukaryote) with bacterial gene ( NPT II gene ) * NOS = nopaline opiant synthase * protein coding region is responsible for resistance to Kanaycin, coming from bacteria * Why we can use a protein coding region from a bacteria and has it being expressed by eukaryotic promoter? Why does the protein coding region give rise to the protein of interest? Ans. Genetic code is universal. It works in both eukaryote and prokaryote. 3. Put the chimeric gene into plant cell * plant cell is now able to grow in the presence Kanamycin * If we put the chimeric gene into the T-DNA, we are able to select the plant that contains the antibiotic gene in the presence of Kanmycin Question of the Day: Why does Kanamycin, an inhibitor of prokaryotic protein synthesis, inhibit plant cell growth?
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Plant have both mitochondria and chloroplast, which were once a parkaryote in the past. (ancient endosymbiont, have bacterial regulatory sequences on their genes). Kanamycin affect these two organelles and plant cells won't be able to generate energy. Regenerate plants:
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Lecture 9 - PLB 113 Lecture 9 III Gene Transfer and...

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