Isolation and characterisation of legumin promoter sequence from chickpea (<i>Cicer arietinum </i>L.)

363-372Seed specific promoters are useful for expression of foreign genes in the seeds. We have isolated a Cicer arietinum legumin promoter from λEMBL genomic library and subcloned in pBluescript II KS (-) in Eco RV and Pst I site. The 2.762 kb Hind II Pst I fragment was sequenced completely by dideoxy chain termination method by creating a set of unidirectional deletions of the inserts in pAKKIII. The insert contains mainly upstream sequence (2240 bps) and only a part of structural gene (522 bps) sequence. The 522 bps of the structural gene shows ~ 80% homology with pea legumin A and this is almost the same as chickpea legumin in its sequence. The amino acid sequence derived from the part of the structural gene was similar to the chickpea 5' part of the legumin structural gene with a few variations. A 21 amino acid signal peptide was also deduced like many other legumes. Transcription start site (CAT) was located at 55 bp upstream of the initiation codon ATG. One codon downstream to ATG codon Hind III site was present. TATA box was observed at- 30 position, with a consensus of CCTATAAATAACC. The consensus CATGCAAG, a part of legumin box was noticed at -110 bp position. At - 295 to -265 bp upstream AGGA box like sequences were observed and a 56 bp perfect repeat was located between - 913 bp and - 972 bp. Strong homology with pea promoter sequence near the CAT sequence was noticed which gradually decreased towards the upstream region. Thus the cloned fragment contains a full length promoter which can be utilised for expression of foreign genes in seeds of chickpea

Improved and convenient method of RNA isolation from polyphenols and polysaccharide rich plant tissues

842-845It has been difficult to extract a good quality total RNA from the plant parts (such as seeds) which contain high levels of phenolic compounds, carbohydrates and other compounds that bind and/or co-precipitate with RNA. A simple, rapid and efficient method for isolating total RNA from polyphenols and polysaccharide rich plant tissues has been developed. Seeds of leguminosae family were chosen for the study. The good quality and high yield of total RNA was achieved with A260/A280 ratio of 1.9. Seeds of three different crops (Cajanus cajan, Dolichos biflorus and Vigna mungo) at different developmental stages were evaluated for total RNA extraction using standardized protocol. Seeds at 21 days after flowering (DAF) gave the best results among others (7 DAF and dry seeds). Quality of isolated RNA from all the three crops was further checked by cDNA synthesis. The extracted RNA was found suitable for further molecular applications such as reverse transcription and cDNA library construction