This study examines the potential for increasing natural L-DOPA drug biosynthesis in Mucuna pruriens by silencing or “knocking down” expression of putative DOPA/tyrosine decarboxylase (Mp-ty/ddc) in situ. Mp-ty/ddc codes for DOPA/tyrosine decarboxylase (Mp-TY/DDC) which converts L-DOPA to dopamine in plants. The hypothesis of the work was that silencing the Mp-ty/ddc gene would result in accumulation of L-DOPA in the plant tissues. This work involved isolation and characterisation of 1.73 kb putative full-length ORF of Mp-ty/ddc. The gene showed 74% homology with TY/DDC protein alignments of other plants in the same taxa, although no enzyme activity was detected when the gene product was heterologously expressed. In addition, a protocol was developed for Agrobacterium mediated transformation of M. pruriens so as to be able to manipulate expression of the DOPA genes in situ. The cotyledonary nodal and hypocotyl tip explants regenerated shoots on M.S media supplemented with 50 μM BA, 0.5 μM NAA and 50 mg l-1 kanamycin selection also the nptII transgene was detected by PCR. The Agrobacteria strains GV3101 harbouring a pGREEN vector and carrying an Mp-ty/ddc antisense were used for the plant transformation experiments. Further work showed that the Mp-ty/ddc gene copy number was 1, the gene expression was highest in roots and stems, followed by seeds and was very low in leaves. On the other hand, L-DOPA-content in seeds was 17-fold higher relative to leaves and 15 fold relative to stems and roots.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:637141 |
| Date | January 2014 |
| Creators | Cabral, Kibedi |
| Publisher | Cardiff University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://orca.cf.ac.uk/70680/ |
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