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Multigene engineering in plants

 Majority of agronomic traits are quantitative and are
 controlled polygenetically.
 Multigene transfer enables –
            1) manipulate entire metabolic pathways
            2) express multimeric proteins
            3) study complex genetic control circuits
 Methods for multigene engineering
            Nuclear genome transformation
            chloroplast genome transformation
Methods for nuclear gene
                        transformation


A.     Crossing Transgenic Lines

 Homozygous transgenic parents are used.


 Eg:Reconstruction of bacterial enzymatic pathway of organic
     mercury detoxification.
               S.P. Bizily et al ,.( 2000)
B.Sequential Transformation


 Is the repetitive insertion of transgenes into a plant.


 E.g. The restoration of fertility in transgenic male sterile
    tobacco by anti sense RNA system.
                     Hird et al .,( 2000)
.
C. Co transformation


Linked Genes                     Unlinked Genes
 Agrobacterium mediated
                                  Agrobacterium - standard
                                   binary vectors
 Direct transfer


 Assisted direct transfer
                                  Direct transfer- conventional
                                   vectors
 Artificial plant chromosomes


 Operon system
                                          Naqvi et al ., (2010)
Chloroplast Transformation

                  Size-120-160kb
                  ~150 genes
                  High copy number.

                  Basic set of genes
                  t RNA
                  r RNA
                  Some ribosomal proteins
                  Polypeptide components of
                    photosystem.
                  8 large sub units of RUBISCO
                  4 sub units of chloroplast
                    specific RNA polymerase.
Multigene engineering in plants
Site Specific Integration By Homologous
               Recombination



                            No Positon Effect


                            No Vector Sequence



                            No Gene Scilencing




                          Bock ( 2007 )
Maternal inheritance and gene containment

                         Genetically engineering crop plants for disease

                           resistance via the chloroplast genome is
                           desirable to achieve high levels of expression
                           and to prevent pollen-mediated escape of
                           transgenes.

                                Daniell et al, .(2001)



 lack of toxicity of transgenic pollen to non-target insects
High Expression & Normal Growth


 Compartmentalization help to protect from proteolytic enzymes.

 Purification is also easy



     No pleiotropic effects




                                      Daniell et al.,(2003)
Integration Of Transgenes In Chloroplast Genome


 Hetero Plasmic Chloroplast - A mix of wild type & transformed

  chloroplast genome.


       1) Inter plastidic
        2) Intraplastidic


• Homoplastomy- All chloroplasts are wild type or transformed.
Integration Of Transgenes In Chloroplast Genome
Application Of Multigene Engineering Through Nuclear
                        Genome


 In Metabolic Engineering

           Tailoring a metabolic pathway

          Nutritional value enhancement

          For dissecting metabolic pathways

          Modification of endogenous pathways

          To synthesize a novel compound or
           macromolecule
Metabolic engineering of carotenoid pathway



                            Carotenoids are secondary
                             metabolites .
                            They are also required by
                             animals as metabolic
                             precursors and antioxidants
                            Some of them are
                             precursors of pro-vitamin A
Golden rice/provit rice




 To combat vit A deficiency.




 The transgenic concept
      introduce , under endosperm –specific
  regulation all genes necessary to establish the
  biochemical pathway for beta carotene , the
  most effective precursor to provit A.
3 genes engineered in SGR-1
                                           IPP

                          Geranylgeranyl diphosphate
                    Daffodil gene-psy              Phytoene synthase

                                        Phytoene
  Vitamin A                                        Phytoene desaturase
   Pathway     Single bacterial gene;-crtl
                performs both functions            ξ-carotene desaturase
 is complete
and functional                          Lycopene
                   Daffodil gene-lcy               Lycopene-beta-cyclase

                                     -carotene
                              (vitamin A precursor)

                                                   Potrykus et al,.(2000)
Carotenoid content in golden rice


  Golden Rice 1 produced 1.6ug/g of total
   carotenoids

  Golden Rice 2 produces 37ug/g of total
   carotenoids (31ug/g of beta-carotene)
Development of super nutritious maize
     4 genes for the vitamins β-carotene, ascorbate, and folate
    transferred to maize genome

     β-carotene:
               Psy1 : corn (Zea mays) phytoene synthase
               crtI: Pantoea ananatis gene encoding carotene
                desaturase

     ascorbate
               dhar: rice dehydroascorbate reductase

     folate
                GCH1: E. coli folE gene encoding GTP cyclohydrolase
                                                      Capell et al.,(2009)
 In canola, 7 transgenes were introduced using an
Agrobacterium mediated linked cotransformation strategy.

        idi ,crtE,crtB, crtI, crt Y, crt Z, crt W   genes from 3 sp:

        Carotenoid     content was 657 µg/g of seed

 In potato, 3 transgenes introduced .
          crtI, crt B, crt Y
Quality Improvement In Oilseeds

                Reconstruction of PUFA pathways
               o Poly unsaturated fatty acid


                     FA with multiple double bond


                     Aliphatic chain with >20C


               • Play major role in human health.


               • 2 classes-w6 & w3
LC-PUFA biosynthesis
pathway
Recent Development In PUFA Pathway Engineering




                                 Sayanova,o et al.,(2009)
Constraints in pathway engineering of PUFA




                               Napier.A(2007)
Approaches to overcome the bottlenecks in LC-PUFA yields


 Identification of improved desaturases


 Identification of specific acyl-exchange mechanisms


 Controlling the flux of LC-PUFAs into TAGs


                                               Napier et al,(.2007)
Impart resistance to biotic and abiotic stresses


 Over expression of bacterial operon impart resistance to lepidopteran
  pests.

        Bt cry2Aa2   operon is transferred to tobacco plants resulted
           in the accumulation of Cry2Aa2 protein at 46.1% of tsp


 Expression of an antimicrobial peptide, MSI-99 as a dicistron in

  transgenic chloroplasts.

                                            Daniell et al, .(2001)
The integration of a yeast trehalose-6 phosphate synthase (TPS) gene

as a dicistron in transgenic chloroplasts was shown to confer drought

tolerance
                                           Lee et al ,.(2003)
PHB production through multi gene engineering
Hyper expression of foreign protein



               Over expression of the B cry2Aa2
                 operon in chloroplasts leads to
                 formation of insecticidal crystals.




               Miller et al., Nat Biot. 2001. 19:71-4
Operon expression & crystal formation


 Bt cry2Aa2 operon(4kb)
  is used .

 Vector used is pLD CtV2


 Foreign gene integration
  determined by PCR
  screening.
Southern blot analysis of   cry2Aa2 protein expression
To & T1 generation          &quantification
INSECT BIOASSAY
           Foreign protein accumulated at
            45.3% of the total soluble protein.

           Insects that are normally difficult
            to control (10-day old cotton
            bollworm, beet armyworm) were
            killed 100% after consuming
            transgenic leaves
Multigene engineering in plants
Thank you…

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Multigene engineering in plants

  • 2. Multigene engineering in plants  Majority of agronomic traits are quantitative and are controlled polygenetically.  Multigene transfer enables – 1) manipulate entire metabolic pathways 2) express multimeric proteins 3) study complex genetic control circuits  Methods for multigene engineering Nuclear genome transformation chloroplast genome transformation
  • 3. Methods for nuclear gene transformation A. Crossing Transgenic Lines  Homozygous transgenic parents are used.  Eg:Reconstruction of bacterial enzymatic pathway of organic mercury detoxification. S.P. Bizily et al ,.( 2000)
  • 4. B.Sequential Transformation  Is the repetitive insertion of transgenes into a plant.  E.g. The restoration of fertility in transgenic male sterile tobacco by anti sense RNA system. Hird et al .,( 2000) .
  • 5. C. Co transformation Linked Genes Unlinked Genes  Agrobacterium mediated  Agrobacterium - standard binary vectors  Direct transfer  Assisted direct transfer  Direct transfer- conventional vectors  Artificial plant chromosomes  Operon system Naqvi et al ., (2010)
  • 6. Chloroplast Transformation  Size-120-160kb  ~150 genes  High copy number.  Basic set of genes  t RNA  r RNA  Some ribosomal proteins  Polypeptide components of photosystem.  8 large sub units of RUBISCO  4 sub units of chloroplast specific RNA polymerase.
  • 8. Site Specific Integration By Homologous Recombination  No Positon Effect  No Vector Sequence  No Gene Scilencing Bock ( 2007 )
  • 9. Maternal inheritance and gene containment  Genetically engineering crop plants for disease resistance via the chloroplast genome is desirable to achieve high levels of expression and to prevent pollen-mediated escape of transgenes. Daniell et al, .(2001)  lack of toxicity of transgenic pollen to non-target insects
  • 10. High Expression & Normal Growth  Compartmentalization help to protect from proteolytic enzymes.  Purification is also easy  No pleiotropic effects Daniell et al.,(2003)
  • 11. Integration Of Transgenes In Chloroplast Genome  Hetero Plasmic Chloroplast - A mix of wild type & transformed chloroplast genome. 1) Inter plastidic 2) Intraplastidic • Homoplastomy- All chloroplasts are wild type or transformed.
  • 12. Integration Of Transgenes In Chloroplast Genome
  • 13. Application Of Multigene Engineering Through Nuclear Genome  In Metabolic Engineering  Tailoring a metabolic pathway Nutritional value enhancement For dissecting metabolic pathways Modification of endogenous pathways To synthesize a novel compound or macromolecule
  • 14. Metabolic engineering of carotenoid pathway  Carotenoids are secondary metabolites .  They are also required by animals as metabolic precursors and antioxidants  Some of them are precursors of pro-vitamin A
  • 15. Golden rice/provit rice  To combat vit A deficiency.  The transgenic concept introduce , under endosperm –specific regulation all genes necessary to establish the biochemical pathway for beta carotene , the most effective precursor to provit A.
  • 16. 3 genes engineered in SGR-1 IPP Geranylgeranyl diphosphate Daffodil gene-psy Phytoene synthase Phytoene Vitamin A Phytoene desaturase Pathway Single bacterial gene;-crtl performs both functions ξ-carotene desaturase is complete and functional Lycopene Daffodil gene-lcy Lycopene-beta-cyclase -carotene (vitamin A precursor) Potrykus et al,.(2000)
  • 17. Carotenoid content in golden rice  Golden Rice 1 produced 1.6ug/g of total carotenoids  Golden Rice 2 produces 37ug/g of total carotenoids (31ug/g of beta-carotene)
  • 18. Development of super nutritious maize  4 genes for the vitamins β-carotene, ascorbate, and folate transferred to maize genome  β-carotene: Psy1 : corn (Zea mays) phytoene synthase crtI: Pantoea ananatis gene encoding carotene desaturase  ascorbate dhar: rice dehydroascorbate reductase  folate  GCH1: E. coli folE gene encoding GTP cyclohydrolase Capell et al.,(2009)
  • 19.  In canola, 7 transgenes were introduced using an Agrobacterium mediated linked cotransformation strategy.  idi ,crtE,crtB, crtI, crt Y, crt Z, crt W genes from 3 sp:  Carotenoid content was 657 µg/g of seed  In potato, 3 transgenes introduced .  crtI, crt B, crt Y
  • 20. Quality Improvement In Oilseeds  Reconstruction of PUFA pathways o Poly unsaturated fatty acid  FA with multiple double bond  Aliphatic chain with >20C • Play major role in human health. • 2 classes-w6 & w3
  • 22. Recent Development In PUFA Pathway Engineering Sayanova,o et al.,(2009)
  • 23. Constraints in pathway engineering of PUFA Napier.A(2007)
  • 24. Approaches to overcome the bottlenecks in LC-PUFA yields  Identification of improved desaturases  Identification of specific acyl-exchange mechanisms  Controlling the flux of LC-PUFAs into TAGs Napier et al,(.2007)
  • 25. Impart resistance to biotic and abiotic stresses  Over expression of bacterial operon impart resistance to lepidopteran pests.  Bt cry2Aa2 operon is transferred to tobacco plants resulted in the accumulation of Cry2Aa2 protein at 46.1% of tsp  Expression of an antimicrobial peptide, MSI-99 as a dicistron in transgenic chloroplasts. Daniell et al, .(2001)
  • 26. The integration of a yeast trehalose-6 phosphate synthase (TPS) gene as a dicistron in transgenic chloroplasts was shown to confer drought tolerance Lee et al ,.(2003)
  • 27. PHB production through multi gene engineering
  • 28. Hyper expression of foreign protein Over expression of the B cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals. Miller et al., Nat Biot. 2001. 19:71-4
  • 29. Operon expression & crystal formation  Bt cry2Aa2 operon(4kb) is used .  Vector used is pLD CtV2  Foreign gene integration determined by PCR screening.
  • 30. Southern blot analysis of cry2Aa2 protein expression To & T1 generation &quantification
  • 31. INSECT BIOASSAY  Foreign protein accumulated at 45.3% of the total soluble protein.  Insects that are normally difficult to control (10-day old cotton bollworm, beet armyworm) were killed 100% after consuming transgenic leaves