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Accueil du site → Doctorat → Namibie → Isolation and characterization of starch, starch biosynthetic genes and protease inhibitors from Marama beab (Tylosema Esculentum)

University of Namibia (2014)

Isolation and characterization of starch, starch biosynthetic genes and protease inhibitors from Marama beab (Tylosema Esculentum)

Nepolo, Emmanuel

Titre : Isolation and characterization of starch, starch biosynthetic genes and protease inhibitors from Marama beab (Tylosema Esculentum)

Auteur : Nepolo, Emmanuel

Université de soutenance : University of Namibia

Grade : Doctor of Philosophy. (PhD) 2014

Marama bean (Tylosema esculentum) is a highly nutritious plant and is currently regarded as a prospective crop for the future in arid zone agri-ecologies of the world. Starch is a major storage component in higher plants and in marama bean it’s mostly found stored in the tuber. Starch is used in both food and non-food industries. Starch biosynthesis involves groups of committed enzymes. Aims of the present study were to determine the physicochemical and pasting properties of native marama bean starch isolate and characterize marama starch biosynthesis genes and detect serine protease inhibitor activities in green and mature marama seeds. The total starch content of marama bean tubers was determined by amyloglucosidase/α-amylase enzymatic digestion and amylose content by Concanavalin A precipitation. A complementary Deoxyribonucleic acid (cDNA) library was constructed from marama tuber for the screening and isolation of Soluble Starch Synthase I (SSSI) and a Polymersae Chain Reaction (PCR) based strategy was used to isolate Adenosine diphosphate-glucose pyrophosphorylase (AGPase) and Starch Branching Enzymes (SBEs) using degenerative primers designed at the conserved motif of corresponding cloned plant starch synthesizing genes. Detection of serine protease inhibitor activities in green and mature marama seeds was established using the reverse zymogram technique and fluorogenic substrate N-alpha-benzoyl-l-arginine 7-amido-4-methylcoumarin hydrochloride and cDNA clone encoding a serine protease gene from marama was isolated using newly developed degenerate PCR primers. Native marama starch content was 87.38 mg starch/gram fresh weight and the total amylose content was 35 %. Phosphate at the C-6 position determined as Glucose-6-Phosphate was 0.788 nmol G6P/mg. The starch granules were round to elliptical with smooth surfaces and their sizes ranged from 8 -20 μm. The pasting properties of pasting temperature, host paste, peak, final viscosity, breakdown and set back showed higher values for marama starch in contrast to commercial potato starch. A cDNA clone encoding a SSSI from T. esculentum was isolated and identified by cDNA screening. The cDNA clone is 684 bp in length and encodes 228 amino acid residues. Sequencing of cloned cDNA showed 100% identity with potato SSSI.The phylogenetic tree indicated the divergence of SSI in higher plants proceed in line with evolutions of monocots from dicots. AGPase and SBEs genomic clones from T.esculentum were isolated and their sequence features revealed. AGPase small and large subunit clones both showed 96% identities with Glycine max, while SBEI and SBEII clones showed 91 and 93% identities to Cicer arietinum and Medicago truncatula. Phylogenetic trees for both AGPase and SBEs clones exhibited higher sequence similarity to the AGPase and SBEs of dicotyledons. Reverse zymogram analysis revealed four putative serine inhibitor activities in mature seeds and not in green seeds. Fluorogenic substrate analysis showed significantly higher (P <0.05) trypsin activity in mature marama bean (2326±356 Fluorescence Units mg 1 protein) than green seeds (362±73 FU mg 1 protein).The analyses showed lower (P <0.05) serine protease activity in both cowpea (877±138 FU mg 1 protein) and soybean (381±36 FU mg 1 protein). A partial cDNA clone encoding a serine protease gene from marama bean showed 100% identity to trypsin inhibitor for cowpea. Physicochemical properties of marama starch revealed considerable high amylose content and pasting properties when compared to other tuberous starch producing crops. Molecular features of starch synthesizing genes in marama were similar to that of the same genes characterized in other plant species. Serine inhibitors activities in marama seed may as well be an indicative that marama seed may have potent serine protease inhibitors which could be utilized in pharmaceuticals and in human health. Physicochemical properties of marama starch would suggest that it could find applications in foods and non-food uses that require higher amylose content and high pasting properties. Isolation and characterization of marama starch biosynthetic genes provide theoretical foundation in understanding the structure and functions of SSSI, AGPase and SBEs. Serine protease inhibitor gene isolation and detection of serine protease inhibitor of marama seeds will broaden the pool for plant serine protease genes and might be an ideal choice for developing pharmaceutical products. Future studies need to establish the potential uses of marama starch, characterize marama starch biosynthetic gene structures, functions, regulation and expression. It would be interesting to study in the future any reason for the higher serine protease activity in mature seeds than in green seeds as established recently.This study has clearly contributed to starch biology by making known for the first time the physicochemical and functional properties of marama tuber starch. It also established for the first time the genetic signature of genes involved in starch biosynthesis of marama bean, which may confer unique properties to marama tuber starch. At the same time, the presence of serine protease inhibitors activities and serine protease inhibitor gene in marama seeds with possible application in pharamaceutical products were investigated for the first time making this investigation novel.

Mots clés : Tylosema esculentum — Marama — Starch biosynthesis — SSSI — AGPase — SBEs — Serine protease inhibitors — Beans, Namibia — Plant genetics, Namibia

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Page publiée le 12 juillet 2014, mise à jour le 7 novembre 2018