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Title: Curcin proteins of biodiesel crop (Jatropha curcas L.): Cytotoxic effects of the crude curcin extract, and molecular approaches for isolation of curcin genes
Authors: Kumar, Nitin
Supervisor: Das, N.
Keywords: Jatropha curcas L.;Curcins;Cytotoxic effects;Sequence analyses;Oligonucleotide primers;Amplicons;DBT
Issue Date: 14-Aug-2015
Abstract: As of now, Jatropha (Jatropha curcas L.) stands as the best solution to the problems associated with production of biofuels, as it is well adapted to marginal soil with low nutrient content, and also suitable in different types of soil conditions, rainfall and climates. Disadvantages of Jatropha are the presence of toxic substances, particularly in the seeds, like curcin and phorbol esters which limits their applications. According to literature study, curcin protein has cytotoxic activity on cancer cell lines. Therefore, one of the objectives was to study the cytotoxic effects of curcin using crude curcin extracts. Here we chose four accessions of Jatropha curcas namely TJS17#03, TJS42#04, TJS35#01 and TJS01#03. First, quality and quantity of seed oils were checked; and oil content was found to be maximum in case of TJS42#05; whereas, free fatty acid content (FFA) content was found to be less in TJS17#03. Based on the available protocol, crude curcin extract was made from the seed kernel of TJS17#03; then its cytotoxic effect was assessed by MTT assay on RAW and HeLa cell lines. The crude curcin extract exhibited inhibitory effect on the cancer cell lines significantly. BLAST analyses revealed that the promoter region of the curcin gene showed considerable sequence divergence if compared with the other members of this family. Multiple sequence alignment was made using different forms of curcin namely curcin precursor, curcin2A, curcin-L precursor and ribosome inactivating protein (RIP). This exercise revealed both sequence identity and divergence between them. Good quality genomic DNA was isolated from field-grown tender leaves using a simple and efficient protocol as introduced in our laboratory. Quality and quantity of the genomic DNA preparations were checked by spectrophotometric analyses. Some forward and reverse primers were made based on the Curcin2A gene sequence as available in the database. Most of the forward primers correspond to the promoter region, and the reverse primers are from the coding regions. The purpose was to amplify the curcin genes with varying upstream (promoter) regions. PCR were carried out using the specific primer pairs. The amplicons corresponding to the individual PCR were noted in order to analyse and compare between the Jatropha accessions. Apart from the expected sizes, more amplicons of varying sizes were also found. All the promising amplicons i.e., PCR products as reported in the study need to be further studied at molecular level.
Description: MT, DBT
Appears in Collections:Masters Theses@DBT

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