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GENETIC ENGINEERING OF NITROGEN FIXATION

GENETIC ENGINEERING OF NITROGEN FIXATION. REPRESENTED BY NIVEDITA CHOUDHARY B.Sc. 2 nd year. www.powerpointpresentationon.blogspot.com. Nitrogen Fixation-.

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GENETIC ENGINEERING OF NITROGEN FIXATION

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  1. GENETIC ENGINEERING OF NITROGEN FIXATION REPRESENTED BY NIVEDITA CHOUDHARY B.Sc. 2nd year www.powerpointpresentationon.blogspot.com

  2. Nitrogen Fixation- • Nitrogen fixation is the process by which nitrogen is taken from atmosphere and converted into nitrogen compounds. • Biological nitrogen fixation was discovered by the Dutch microbiologist Martinus Beigerinck. • Natural processes fix about 190×1012 g yr-1 of nitrogen through the following processes-

  3. Types of nitrogen fixation • Lightning 2.Photochemical Reaction 3.Industrial Nitrogen Fixation 4.Biological Nitrogen Fixation

  4. Types of Biological Nitrogen Fixation • Symbiotic Nitrogen Fixation • Nonsymbiotic Nitrogen Fixation • Symbiotic Nitrogen Fixation- • In symbiotic nitrogen fixation the plant and bacteria depend on each other. • Rates of nitrogen fixation are highly variable and are dependent on the bacterial strain-legume cultivar used, soil and other environmental conditions. • 100kg of nitrogen are reported to be derived from symbiotic nitrogen fixation.

  5. Continue……………… • The highest amount of nitrogen fixed by the legumes takes place either the flowering stage or during pod fill stage. • Legume symbioses- eg- rhizobium species • Associations with Frankia- Frankia is a group termed Actinomycetes-filamentous bacteria that are noted for their production of air-borne spores. They form nitrogen fixing root nodules with several woody plants.eg-elder,sea buckthorn. • Cyanobacterial associations-The photosynthetic cyanobacteria often live as free-living organisms in pioneer habitats such as desert soils or as symbionts with lichens in other pioneer habitats.they form nitrgen fixing nodulation.

  6. Nonsymbiotic nitrogen fixation • In nonsymbiotic nitrogen fixation the bacteria not associated with plant but they fix nitrogen into the plant. • Azotobacter and Azospirillum fix nitrogen into the plant by the nonsymbiotic process. • Azospirillum is not only capable of nitrogen fixation but also codes for plant growth hormones auxins and cytokinins. • These bacteria are the free living bacteria that fix nitrogen.

  7. Mechanism of Nitrogen fixation • The biological process of nitrogen fixation is catalyzed by the nitrogenase enzyme, an enzyme complex containing the nitrogenase reductase and dinitrogenase. • Nif genes forming a gene cluster of 24 kb nucleotides which are located between the genes encoding for histidine and shikimic acid. • The cluster is organized in 7 operons i.e. transcription units (QB AL FM VSUX NE YKDH J). Nif HDKY operon encodes nitrogenase.

  8. Continue………… • Nitrogenase contains the two proteins molybdoferredoxin and azoferredoxin. • In most bacteria electrons are passed from NAD(P)H or pyruvate to ferredoxin, a FeS protein. • Azoferredoxin transfers electrons from reduced flavodoxin (or ferredoxin) to molybdoferredoxin. Molybdoferredoxin is a α2β2 tetramer. • The alpha and beta subunits are similar but distinct and are encoded by genes nifK and nifD. Each tetramer contains 2 Mo and several FeS groups. • Azoferredoxin is a dimer of identical subunits encoded by nifH and contains a single Fe4S4 group per dimer. In

  9. Nodulation • legumes release compounds called flavonoids from their roots, which trigger the production of nod factors by the bacteria. when the nod factor is sensed by the root, number of biochemical and morphological changes are occur: 1. cell division is triggered in the root to create the nodule. 2. the root hair growth is redirected to wind around the bacteria multiple times until it fully encapsulates one or more bacteria. • Legumes and actinorhizal plants regulate gas permeability in their nodules, maintaining a level of oxygen within the nodule. • A nodule contains an oxygen-binding heme protein called leghemoglobin that gives the nodules a pink color.

  10. Steps involved in nodulation- • Attachment of bacteria to the roots of higher plant- 1.Lectin-mediated root hair binding- • The host-microsymbiont specificity is governed by a specific plant protein called lactins involved in recognition of compatible symbiont. It is supplemented by secretion of specific polysaccharides by the symbiont termed as callose which helps in attachment of rhizobia on host root surface. • The rhizobial cell that infect clover posses the cross reactive antigen(CRA).clover roots absorb CRA and infective R.trifolii cells.

  11. Continuous…… 2. Recadhesin -mediated root hair binding- • A Ca2+ binding bacterial protein called rhicadhesin also appears to be involved in bacterial attachment to legume root hairs. • Calcium ions are not involved in binding of rhicadhesin to the root surface. calcium ions appears to be involved in anchoring rhicadhesin to the rhizobial cell surface. • An R.l. bv. Trifolii-adheringprotein,called RapA1,and R.l.bv.viciae rhicadhesin both are secreted proteins that bind calcium, bind at bacterial cell poles and to root hairs, and mediate calcium dependent agglutination.

  12. Gene involved in nitrogen fixation- • Nod genes- plant root produce various type of flavonoids that stimulates the release of Nod factor by Rhizobium. • The eight nod genes present in the Rhizobium bacteria. These nod genes are nod A,B,C,D,E,F,I, and nod J. • nodD genes regulates the transcription of other nod genes. • Nif gene – nif gene isolate from k.pneumoniae.these nif genes are nif J,H,D,K,T,Y,E,N,X, U,S,V,W,Z,M,F, L, A, B, and nifQ. • Nif A and nif L gene regulate the other nif genes.

  13. Phytohormones that involve in nodule formation • Two processes infection and nodule organogenesis-occur simultaneously during root nodule formation. • During the infection processrhizobia that are attached to the root hairs release Nod factors that induce a pronounced curling of the root hair cells. • The next step is formation of the infection thread, an internal tubular extension of the plasma membrane that is produced by the fusion of Golgi-derived membrane vesicles at the site of infection. • Ethylene is synthesized in the region of the pericycle, diffuses into the cortex, and blocks cell division opposite the phloem poles of the root. ,

  14. Alternative Nitrgen fixation system

  15. Genetic engineering of Rhizobium • The nif and nod gene of the fast growing Rhizobium bacteria are located on plasmid, called symbiotic plasmid. • The rhizobium bacteria have nod gene cluster for nodulation. • These nod genes are nodD,A,B,C,E,F,I, and nodJ . • The nod I gene encodes a proteins that may be involved in membrane transport. • The nod E gene may help to prevent nodulation of legumes other than the normal host for a particular Rhizobium species.

  16. Continues…….. • The last of these nod genes, nod D,has been shown to be regulatory, controlling the transcription both of itself and of the other nod genes in the cluster. • The plant release some flavonoids that stimulates the release of Nod factor by Rhizobium.

  17. Genetic engineering of nitrogen fixation • Nif genes are involved in nitrogen fixation that are isolate from k.pneumoniae and transfer into E.coli. • The Nif gene cluster required for maturation and regulation of the nitrogenase enzyme. • These Nif genes are nif J,H,D,K,T,Y,E,N,X, U,S,V,W,Z,M,F, L, A, B,Q. • NifL and nifA regulate the expression of all the other genes that synthesized the nitrogenase enzyme. • The RNA polymerase bind to the Nif gene promoter for the initiation of transcription.

  18. Continue……………. • The ntrA gene codes for a protein called a sigma factor, that confers on RNA polymerase the ability to recognize the nif gene promoter. • The ntrA gene and three additional genes-glnA,ntrB,and ntrC- are the part of a global system that regulates many aaspects of nitrogen metabolism in bacteria.

  19. Regulation of nif gene-

  20. Engineering of nif gene- • The nif gene cluster is introduced in the chloroplast genome through genetic engineering. • The nif gene does not express without ntrA gene because ntr gene code the sigma protein that required for the recognition of nif gene promoter.

  21. Methods of nif gene transformation into chloroplast genome- • Three methods developed for introducing chloroplast vector back into the organelle genome- • By using Ti plasmid the gene is transferred into plant. • A two step transformation in which a plasmid with both nucleus and chloroplast specific markers is first transformed into the cytoplasm and, having established replication within the organism, selection is made for the chloroplast specific marker on the plasmid which could only be expressed in the plastid. 3. Microinjection method to interduce cloned genes directly into chloroplast. By these processes the nif gene transferred into plants.

  22. THANK YOU

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