Gene Transfer to Plant PDF

SSCG 2713 Genetic Engineering Gene Transfer To Plant Dr. Nazrin Abd Aziz UTM KUALA LUMPUR Methods of delivering DNA into plant cells Biological Agrobacterium Viruses Physical Biolistic gun/Particle bombardment Microinjection Silicon carbide...

SSCG 2713 Genetic Engineering Gene Transfer To Plant Dr. Nazrin Abd Aziz UTM KUALA LUMPUR Methods of delivering DNA into plant cells Biological Agrobacterium Viruses Physical Biolistic gun/Particle bombardment Microinjection Silicon carbide whiskers Carbon nanofibers  Overview Ti plasmid is inserted into bacterium When the bacterium mix with plant cells, the Ti plasmid moves into plant cell and transfer the gene of interest into plant chromosome What is transgene?  Agrobacterium tumefaciens causes crown gall disease of a wide range of dicot plants The disease gains its name from the large tumour-like swellings (galls) This crown gall is caused by A. tumefaciens that harbors Ti plasmid The Ti plasmid contains several genes that can induce the (i) formation of tumour and (ii) associated changes in plant metabolism These genes can be transferred and integrated into the plant’s genomic DNA What are dicot plants? Crown gall formation due to the Agrobacterium infection (Original) Ti plasmid Stand for tumour-inducing plasmid Consist of: 1. T-DNA, 2. vir genes, and 3. gene encoding for opine catabolism Original Ti plasmid that can be found naturally in A. tumefaciens  T-DNA region T-regions are defined by T-DNA border sequences Consist of auxin, cytokinin and opine genes When expressed in plant, auxin and cytokinin induce the formation of tumor Opine will become the nutrient source for the bacteria Original Ti plasmid that can be found naturally in A. tumefaciens What are auxin, cytokinin and opine? Opine Catabolism The region control expression of opine transport and catabolism Opines are derivatives of amino acid or sugar phosphates that can be catabolized to use in the form of nutrients The types of opines found in Ti- plasmid are nopaline and octopine types. Original Ti plasmid that can be found naturally in A. tumefaciens Virulence Region Vir genes located in virulence region play a crucial role in the T-DNA transfer to the plant Virulence region consist of: VirA and VirG VirD1 and VirD2 VirB VirE2 Original Ti plasmid that can be found naturally in A. tumefaciens  https://youtu.be/yesNHd9h8k0?si=JcmhaVNrkbYpr8Pi Molecular Basis of T-DNA Transfer McCullen, C.A., & Binns, A.N. (2006). Agrobacterium tumefaciens and plant cell interactions and activities required for interkingdom macromolecular transfer. Annual review of cell and developmental biology, 22, 101-27.  https://youtu.be/yesNHd9h8k0?si=JcmhaVNrkbYpr8Pi Molecular Basis of T-DNA Transfer 1. VirA and VirG will be activated when receive stimulus from the plant such as acetosyringone and sugar 2. VirA and VirG will activate another vir genes such as VirD1, VirD2, VirB and VirE McCullen, C.A., & Binns, A.N. (2006). Agrobacterium tumefaciens and plant cell interactions and activities required for interkingdom macromolecular transfer. Annual review of cell and developmental biology, 22, 101-27.  https://youtu.be/yesNHd9h8k0?si=JcmhaVNrkbYpr8Pi Molecular Basis of T-DNA Transfer 3. VirD1 and VirD2 will cleave the T-DNA at the right and left border. VirD2 will remain attach at the right border of T- DNA. McCullen, C.A., & Binns, A.N. (2006). Agrobacterium tumefaciens and plant cell interactions and activities required for interkingdom macromolecular transfer. Annual review of cell and developmental biology, 22, 101-27.  https://youtu.be/yesNHd9h8k0?si=JcmhaVNrkbYpr8Pi Molecular Basis of T-DNA Transfer 4. VirB form a membrane channel to export the T-DNA from bacteria to plant McCullen, C.A., & Binns, A.N. (2006). Agrobacterium tumefaciens and plant cell interactions and activities required for interkingdom macromolecular transfer. Annual review of cell and developmental biology, 22, 101-27.  https://youtu.be/yesNHd9h8k0?si=JcmhaVNrkbYpr8Pi Molecular Basis of T-DNA Transfer 5. VirE2 form a pore in the plant cytoplasmic membrane to facilitate the passage of the T- DNA strand. VirE2 also protect the T-DNA from degradation. McCullen, C.A., & Binns, A.N. (2006). Agrobacterium tumefaciens and plant cell interactions and activities required for interkingdom macromolecular transfer. Annual review of cell and developmental biology, 22, 101-27.  https://youtu.be/yesNHd9h8k0?si=JcmhaVNrkbYpr8Pi Molecular Basis of T-DNA Transfer 6. VirD2 that attached to the T- DNA, guide the T-DNA molecule to move to the plant cell via export channel 7. While inside the plant, VirD2 assist the T-DNA to integrate in the plant genome McCullen, C.A., & Binns, A.N. (2006). Agrobacterium tumefaciens and plant cell interactions and activities required for interkingdom macromolecular transfer. Annual review of cell and developmental biology, 22, 101-27. Manipulation of Agrobacterium for genetic engineering purposes 2 approaches in Agrobacterium-mediated gene transfer 1. Co-integration by disarming the T-DNA 2. Binary vector construction 1. Co-integration method Remove the auxin, cytokinin and opine genes, and replace it with our gene of interest Virulence Opine region catabolism Ori 1. Co-integration method Ori 2. Binary vector Using co-integration technique is quite difficult due to some reason: - Large size of Ti plasmid (the larger the plasmid, the lower the transformation efficiency) - Low copy number of Ti plasmid To overcome this, binary system is introduced as alternative Systems in which T-DNA and vir genes are located on separate plasmids (One plasmid contain the T-DNA region, another plasmid contain the vir genes) 2. Binary vector 1. Move T-DNA region from Ti plasmid into a separate plasmid (name: Binary vector plasmid or Disarmed Ti plasmid) 2. Replace the auxin, cytokinin and opine genes in the T-DNA region with our gene of interest 3. Make sure the gene of interest is located between the right and left border in the binary vector plasmid 4. Vir genes are retained on Ti plasmid (or also known as vir helper plasmid) 5. Transform Agrobacterium with both plasmid (co-transformation) 6. Infect plant with transformed bacteria Gene transfer to plant In order to facilitate the Agrobacterium infection, wounding on the plant may be required Plant wounding will release chemicals like acetosyringone and sugar to activate the Vir genes Biolistic gun A physical method to transfer the gene into plants Known as particle gun / particle bombardment Use to shoot a piece of DNA into a recipient cell Biolistic gun - How its work? Tungsten or gold bead are coated with the gene of interest and fired into plant tissue using Helium gas The particle pass through the plant cell, and release the DNA of interest in the plant nucleus In the nucleus, the DNA of interest integrate with plant genome  Parameters https://www.sciencedirect.com/topics/nursing-and-health-professions/biolistic-transformation Material to be coated with the DNA should be inert Appropriate target cell for regeneration oCell/tissue must be competent oConsider using protoplast to increase the rate of penetration Depth of penetration of the gene of interest oLack of penetration may cause the coated DNA cannot reach the nucleus oToo strong penetration may cause the coated DNA penetrate the cell Inert = not reactive, so does not interact with DNA and plant cell Advantage and Disadvantage Advantage Disadvantage Can be used to transfer gene of The intracellular target is interest to all plant species random No living vector required Gene of interest is not protected Protocol relatively simple High cost of equipment and coating material Thank you!