Isolation and characterization of type III polyketide synthases from chethikoduveli (Plumbago rosea L)
By: Dhanya Radhakrishnan.
Contributor(s): P Padmesh Pillai (Guide).
Material type:
BookPublisher: Vellayani Department of Plant Biotechnology, College of Agriculture 2014Description: 112p.Subject(s): Plant BiotechnologyDDC classification: 660.6 Online resources: Click here to access online Dissertation note: MSc(INT) Abstract: The study entitled “Isolation and characterization of type III polyketide synthases from chethikoduveli (Plumbago rosea Linn.)” was conducted at the Biotechnology and Bioinformatics Division, JNTBGRI, Palode,
Thiruvananthapuram, during 2013-2014. The objective of the study was to isolate and sequence characterize type III polyketide synthase gene(s) involved in the biosynthesis of plumbagin in Plumbago rosea. This knowledge would eventually help in augmenting the production of plumbagin through the development of high yielding cell lines. Furthermore, chemical variation with respect to plumbagin content and genetic diversity using ISSR markers was also carried out in available accessions of Plumbago rosea.
Nested degenerate primers specific to conserved regions of previously reported CHSs were used to amplify the cDNA synthesised from the reverse transcribed total RNA isolated from TBG-102. The amplicon was cloned and sequenced. The full length cDNA was elucidated using 5’ and 3’ RLM-RACE followed by cloning and sequencing. The full length cDNA was found to be 1,197 bp coding for 398 amino acids. The protein was calculated to have a molecular mass of 43.5 kDa and a theoretical pI of 5.89. BLASTX analysis of the full length cDNA showed 89 % identity with naringenin chalcone synthase from Hypericum androsemum, chalcone synthase from Hypericum sampsonii and aromatic polyketide synthase from Hypericum hookerianum. Simialr interpretation was drawn from the constructed phylogentic tree.In brief, it is concluded that the isolated full length cDNA belongs to typical CHS family and therefore, additional non-CHS class of PKSs would be involved in plumbagin synthesis.
Genetic diversity analysis with ISSR markers revealed high degree of polymorphism as the primers generated 71 scorable bands, out of which 70 were polymorphic (98.6 % polymorphism). Thereby, the accessions were concluded to have high genetic diversity.
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Chemical analysis in these accessions showed variation in plumbagin content from 0.2 % to 0.7 %. The observed variation in plumbagin content may be due to the prevailing high genetic diversity in these accessions and variation in edaphic factors. The plumbagin content in TBG-102 was estimated using HPLC and spectrophotometric analysis. Both methods produces comparable results.
Therefore cold extraction method was found to be reproducible.
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KAU Central Library, Thrissur Theses | Reference Book | 660.6 DHA/IS (Browse shelf) | Not For Loan | 173447 |
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MSc(INT)
The study entitled “Isolation and characterization of type III polyketide synthases from chethikoduveli (Plumbago rosea Linn.)” was conducted at the Biotechnology and Bioinformatics Division, JNTBGRI, Palode,
Thiruvananthapuram, during 2013-2014. The objective of the study was to isolate and sequence characterize type III polyketide synthase gene(s) involved in the biosynthesis of plumbagin in Plumbago rosea. This knowledge would eventually help in augmenting the production of plumbagin through the development of high yielding cell lines. Furthermore, chemical variation with respect to plumbagin content and genetic diversity using ISSR markers was also carried out in available accessions of Plumbago rosea.
Nested degenerate primers specific to conserved regions of previously reported CHSs were used to amplify the cDNA synthesised from the reverse transcribed total RNA isolated from TBG-102. The amplicon was cloned and sequenced. The full length cDNA was elucidated using 5’ and 3’ RLM-RACE followed by cloning and sequencing. The full length cDNA was found to be 1,197 bp coding for 398 amino acids. The protein was calculated to have a molecular mass of 43.5 kDa and a theoretical pI of 5.89. BLASTX analysis of the full length cDNA showed 89 % identity with naringenin chalcone synthase from Hypericum androsemum, chalcone synthase from Hypericum sampsonii and aromatic polyketide synthase from Hypericum hookerianum. Simialr interpretation was drawn from the constructed phylogentic tree.In brief, it is concluded that the isolated full length cDNA belongs to typical CHS family and therefore, additional non-CHS class of PKSs would be involved in plumbagin synthesis.
Genetic diversity analysis with ISSR markers revealed high degree of polymorphism as the primers generated 71 scorable bands, out of which 70 were polymorphic (98.6 % polymorphism). Thereby, the accessions were concluded to have high genetic diversity.
112
Chemical analysis in these accessions showed variation in plumbagin content from 0.2 % to 0.7 %. The observed variation in plumbagin content may be due to the prevailing high genetic diversity in these accessions and variation in edaphic factors. The plumbagin content in TBG-102 was estimated using HPLC and spectrophotometric analysis. Both methods produces comparable results.
Therefore cold extraction method was found to be reproducible.
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