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Subject: Agricultural Biotechnology × Author: Namdeo, Jadhav Roshankumar × End date: 2017 × Start date: 2010 ×
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    ThesisItemOpen Access
    Molecular cloning, expression and characterization of methionine-rich prolamin seed storage protein geneE (fimP2) of finger millet (Eleusine coracana)
    (G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2017-08) Namdeo, Jadhav Roshankumar; Anil Kumar
    Nutrition is very much important for healthy human being. Content and composition of diet is crucial for preventing protein energy malnutrition. Any significant alteration of nutrients due to improper dietary practices affects the health outcome. Finger millet (Eleusine coracana) is one of the economically and agriculturally important crops of arid and semi arid regions and it possesses high nutritional value. Finger millets can be proposed as nutraceutical crops after identification of quality proteins of high biological value and enriched with essential amino acid (EAA). It can be explored for the development of preventive modalities and food supplements. Amino acids like methionine as an EAA has been suggested as EAA and is one of the four main dietary sources of methyl groups, its deficiency can be associated with fatty liver, atherosclerosis, neurological disorder and tumorigenesis-all are methylation related disorders. Therefore, having methionine rich protein in our diet or taking it as neutraceuticals is one of the important modes of enrichment of our diet. Attempts were made in our laboratory to report for the first time de novo assembly of transcriptome data from two finger millet genotypes differing in their seed protein content. The annotation of the transcriptome data has made it possible to annotate novel and nutritionally important genes. One of such gene fimP2 (finger millet Prolamin-2 encoding gene) was cloned and characterized for its physicochemical properties and enriched with 14.8% methionine amino acid. The fimP2 gene was cloned in bacterial system for heterologous expression. The recombinant protein was further isolated and purified. In silico studies were employed for physico-chemical characterization as well as the structure interpretation of the protein. The biological roles of the protein were also predicted by homology searching, conserved domain analysis etc. The presence of two internal repetitive conserved domain of the protein is suggestive of seed storage protein, indicating its roles in fulfillment of nutritional need. In order to determine the tissue specificity of fimP2 gene, transcript profiling was performed in two genotypes of finger millet differing in seed protein content i.e. GE-1437 and GE-3885. The expression analysis of the gene by real-time PCR showed an increasing order of expression in developing spikes from S1 to S4 however the gene was abundantly expressed in GE-3885-a high protein genotype indicating that higher grain protein content is also linked with higher expression of prolamin protein. The results of present investigation led to identify and characterize a novel FIMP-2 protein envisaged to have nutritional role for serving methyl donor in many biological processes. Thus can be further validated using various nutritional studies and molecular biology approaches to prove its nutraceutical and biological function.