Biogenetics and natural resources Dept.
IDENTIFICATION OF GENES AND GENE-NEAR
REGIONS RELATED TO ACTIVE COMPOUNDS
IN Warburgia ugandensis - AN IMPORTANT
AGROFORES...
Currently 80% of people in sub-Saharan Africa rely on
phytomedicines for health remedies.

Two- thirds of all medicinal pl...
Warburgia Ugandensis
                 Biology

                  The genus Warburgia is one of the five genera of the
    ...
Importance

  Used for treatment of malaria, an economically
 important disease of the tropics.

 Have both antibacterial ...
General ICRAF Objectives on Warburgia study
To develop storage protocols for the otherwise recalcitrant seeds of
Warburgia...
Pilot study on the potential of different tissue extracts from W.
ugandensis trees as anti-malarial. (Dr Geoffrey M. Rukun...
Sesquiterpenes


                 Plant secondary metabolites

                 They include:- Muzigadial,
               ...
Goals of my Ph.D

Elucidate genes involved in sesquiterpene biosynthesis in Warburgia and
thereby identify and develop a m...
Study approach
DNA Approach
 PCR amplification of sesquiterpene genes directly from the genomic DNA using
 degenerate prim...
PCR with Degenerate primers.


 A number of degenerate primers have been planned based on
 conserved regions of sesquiterp...
TECHNOLOGY

                  Methyl filtration.
 Aimed at obtaining gene-near regulatory elements/CpG islands

 Their rol...
Frequency of CpG islands in the isolated regions in rice
                      genome
Results based on the rice genome as ...
Flow diagram

      C C
      G G G G
          C C                       5m C  C G G 5m
                                 ...
T8_Stem
                                                   LADDER




                                                    ...
PCR amplification and validation on Agarose




              _




          +
DNA-Chips (DNA micro-arrays)
 DNA
Hybridization on DNA chip

                             Methylation
                            fragments or
             ...
Scanning


                            Cy3                  Cy5
                           532nm                635nm

   ...
Hybridized and scanned Microarray Chip




                               Statistical analysis of:
                       ...
Progress

DNA Approach
 PCR amplification of sesquiterpene genes directly from the genomic DNA using
 degenerate primers (...
Conclusion and perspectives

New genes possibly involved in sesquiterpene biosynthesis
will be identified and isolated fro...
ACKNOWLEDGEMENTS


ICRAF (Trees and Markets team)
OÄD
ARCSr
THANK YOU
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identification of genes and gene-near regions related to active compounds in Warburgia ugandensis

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identification of genes and gene-near regions related to active compounds in Warburgia ugandensis

  1. 1. Biogenetics and natural resources Dept.
  2. 2. IDENTIFICATION OF GENES AND GENE-NEAR REGIONS RELATED TO ACTIVE COMPOUNDS IN Warburgia ugandensis - AN IMPORTANT AGROFORESTRY TREE - Edward Muge (Ph.D student). - Ramni Jamnadass (ICRAF). - Kornel Burg (ARCSr). - Joseph GlŐssl (BOKU). PICME
  3. 3. Currently 80% of people in sub-Saharan Africa rely on phytomedicines for health remedies. Two- thirds of all medicinal plants are trees and the majority of plant material is harvested unsustainably from the wild. Little is known about the quality and effectiveness of plants that institutions like ICRAF would wish to introduce into farming landscapes. Among the trees targeted for this evaluation is the East African Greenheart (Warburgia ugandensis). Common names:- pepper bark tree, East African greenheart, Ol- msogoni, muthiga, soget.
  4. 4. Warburgia Ugandensis Biology The genus Warburgia is one of the five genera of the family Canellaceae. Has only three species, all African (W. salutaris, W. stuhlmannii, W. ugandensis). Distribution W.salutaris is found in Southern Africa, W.stuhlmannii is endemic to coastal Kenya and Tanzania while W.ugandensis has a wider distribution within Eastern Africa.
  5. 5. Importance Used for treatment of malaria, an economically important disease of the tropics. Have both antibacterial and antifungal medicinal qualities. (Aqueous bark methanolic extracts and unique sesquiterpene 1-4 dialdehydes). Curative of ailments such as stomach-ache, constipation, toothache, common cold, cough, fever, muscle pains, weak joints and measles. The dialdehydes possess potent antifeedant activity against African armyworms. Other minor uses include food, fodder, fuel, timber and gum.
  6. 6. General ICRAF Objectives on Warburgia study To develop storage protocols for the otherwise recalcitrant seeds of Warburgia ugandensis. To asses population density in selected populations across the natural distribution range of the species within the genus Warburgia. Resolve confusion over the present taxonomic status of the members of genus Warburgia Identification and establishment of their population structure and genetic variation within its natural distribution range. To determine the genetic relationship within and between the species of and identify the type of mating system for W. ugandensis. To develop marker system for breeding/selection purposes to obtain genotypes with high anti-malarial effectivity and to further understand the effect of environment on stability of the anti-malarial phenotype.
  7. 7. Pilot study on the potential of different tissue extracts from W. ugandensis trees as anti-malarial. (Dr Geoffrey M. Rukunga (KEMRI). Sample Plant part IC50 Values (µg/ml) Trends. code The roots are by far more active A1-T3 Young leaves >250 than the leaves. A2-T3 Old leaves >250 A3-T3 Root bark 4.09±0.24 T3 roots had the highest activity B1-T5 Young leaves 241.24±0.88 followed by T5, then T6. B2-T5 Old leaves 33.70±2.94 B3-T5 Root bark 4.70±0.35 The work on the roots of T8 and all stem barks is yet to be C1-T6 Young leaves 162.74±43.08 completed. C2-T6 Old leaves 212.74±16.0 C3-T6 Root bark 8.55±0.52 D1-T7 Young leaves 120.21±34.38 D2-T7 Old leaves 131.56±3.98 D3-T7 Root bark 14.88±2.65 E1-T8 Young leaves >250 E2-T8 Old leaves >250 CQ 0.0173 ± 0.0008
  8. 8. Sesquiterpenes Plant secondary metabolites They include:- Muzigadial, Mukaadial, Polygodial, Warburganal e.tc.
  9. 9. Goals of my Ph.D Elucidate genes involved in sesquiterpene biosynthesis in Warburgia and thereby identify and develop a marker system for breeding/selection purposes. This marker would enable identification of Warburgia genotypes with high anti-malarial affectivity using a procedure called CpG island microarrays. Finding genomic regions that are differentially methylated (variation/diversity) in different individuals Isolating gene and gene near sequences correlated to sesquiterpene biosynthesis. Is there any correlation of the identified gene(s) (marker) with altered anti-malarial activity?
  10. 10. Study approach DNA Approach PCR amplification of sesquiterpene genes directly from the genomic DNA using degenerate primers (based on known plant sesquiterpene sequences) Preparing genomic libraries after methyl filtration for comparative purpose among different tissues/plants on micro array RNA Approach RNA has been isolated from the root and stem cambia and leaves of the different genotypes for establishment of cDNA libraries About 1000 cDNA clones from each tissue will be picked and spotted on microarray chip. This array will be comparatively hybridized with RNA probes originating from genotypes representing different anti-malaria efficiency. The clones showing differential expression will be sequenced and annotated by BLAST.
  11. 11. PCR with Degenerate primers. A number of degenerate primers have been planned based on conserved regions of sesquiterpene genes. These are still being tried out on PCR as well as sequencing candidate PCR products. Degenerate house keeping genes primers have yielded positive results. Other techniques including genome walking is also being tried out. Chemical composition profiling has shown that most of the compounds have more relatedness to triterpenes as opposed to sesquiterpenes, Triterpene primers have also been planned.
  12. 12. TECHNOLOGY Methyl filtration. Aimed at obtaining gene-near regulatory elements/CpG islands Their role in gene regulation has been proven both in animals and plants CG/CpG island is a short stretch of DNA in which the frequency of CG sequence is higher than other regions. CpG islands are found near or within genes (introns) Cytosine residues of the DNA are generally methylated in eukaryotes and extensively methylated in plants (CmG, CmCG)
  13. 13. Frequency of CpG islands in the isolated regions in rice genome Results based on the rice genome as model plant Nearly 70 % of enriched clones contained CpG island sequences About 1/3 of the clones originated from transposons Similar results have been obtained in: Sweet Potato Banana Warburgia
  14. 14. Flow diagram C C G G G G C C 5m C C G G 5m C C 5mC G G C 5m G G G G C 5m C 5m C G C G 5m G C 5m G C Digest with HpaII+MspI and ligate adapter C G G C G G C C G G C5m 5mC C G G 5mC G G C C5m G G5mC 5mC G G 5mC C5mC G G C Amplify small adapter-ligated fragments Genomic representation
  15. 15. T8_Stem LADDER T8_Root T8_Leaf Genomic DNA from different tissues of Warburgia ugandensis(T8). KB Material for cloning, colony picking, and 5000 PCR for printing on slides. 2000 800 400 100 Cloning
  16. 16. PCR amplification and validation on Agarose _ +
  17. 17. DNA-Chips (DNA micro-arrays) DNA
  18. 18. Hybridization on DNA chip Methylation fragments or cDNA Mix eg. Root + Stem
  19. 19. Scanning Cy3 Cy5 532nm 635nm Detection Emisssion Excitation Relative Abundance Cy3 / Cy5 (ratio) 1 2 3 Spot (gene)
  20. 20. Hybridized and scanned Microarray Chip Statistical analysis of: changes in gene expression or methylation
  21. 21. Progress DNA Approach PCR amplification of sesquiterpene genes directly from the genomic DNA using degenerate primers (based on known plant sesquiterpene sequences) Completed genomic libraries after methyl filtration and spotted the clones on microarray chips. Began initial hybridization standardizations and data analysis. RNA Approach RNA has been isolated from the root and stem cambia and leaves of the different genotypes for establishment of cDNA libraries. Completed cDNA library from leave tissue and picked 10,000 clones for spotting.
  22. 22. Conclusion and perspectives New genes possibly involved in sesquiterpene biosynthesis will be identified and isolated from yet poorly studied plant. The research will bring first sequence data on W. ugandensis. Partial sequences from some house keeping genes have so far been obtained. The relation of this particular secondary metabolites type to anti malarial properties is expected to be clarified. Comparative evaluation of the results obtained both in the DNA based and in the RNA based approach will then be used to try and identify genes involved in sesquiterpene biosynthesis.
  23. 23. ACKNOWLEDGEMENTS ICRAF (Trees and Markets team) OÄD ARCSr
  24. 24. THANK YOU

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