Curcin gene promoters in Jatropha (Jatropha curcas L.): sequence analysis, prediction of cis-regulatory motifs, and studies on amplicon profile

Abstract

Jatropha (Jatropha curcas L.) as a renewable energy source is the best solution to the problems associated with production of biofuels, as it is very easy to grow. It is a multipurpose shrub of significant economic importance because of its several potential industrial and medicinal uses. Apart from a number of advantages, presence of toxic substances particularly in the seeds, like curcin and phorbol esters limits its application. Here we chose eight accessions of Jatropha curcas namely TJS 17#03, TJS 42 #04, TJS 35#01, TJS 19#17, TJS 06#24 , TJS 01#03, TJS 01#04 and TJS 46#04. Our work is mainly restricted to the promoter region of the curcin2A gene of Jatropha curcas. First, Quality and quantity of the genomic DNA preparations were checked by nanodrop spectrophotometry. BLAST and multiple sequence alignment 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. PCR were carried out using the specific primer pairs. 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 region. The purpose was to amplify the curcin genes with varying upstream (promoter) regions. The amplicons corresponding to the individual PCR were noted in order to analyse and compare between the Jatropha accessions. Apart from the expected sizes, some 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.