Characterization and expression of myo-inositol- 3- phosphate synthase (MIPS) gene in Glycine max

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dc.contributor.advisorArchana Sachdev
dc.contributor.authorSWATI KUMARI
dc.date.accessioned2016-08-19T13:39:09Z
dc.date.available2016-08-19T13:39:09Z
dc.date.issued2013
dc.descriptiont-8793en_US
dc.description.abstractD-myo-inositol-3-phosphate synthase (EC 5.5.1.4; MIPS) is the only isomerase that catalyzes the conversion of glucose-6-phosphate to D-myo-inositol-3-phosphate, a sole synthetic source of myo-inositol. Phytic acid (myo-inositol-1,2,3,4,5,6- hexakisphosphate) which is the principal storage form of phosphorus (60-80%) in plant seeds, is further generated by a stepwise phosphorylation of myo-inositol. Poorly digested by monogasterics as it chelates essential mineral cations and proteins thereby reducing their bioavailability, classifying it as an anti-nutrient. In soybean, transcripts encoding MIPS1 are expressed early during the cotyledonary stage of seed development to function in phytic acid biosynthesis. In the present study, we report the cloning and characterization of the MIPS1 gene from developing seeds of soybean (GmMIPS1). A full-length GmMIPS1 cDNA (Glycine max L. Merr.) of 1,791 bp, containing an ORF of 1,533 bp, encoding 510 amino acids was cloned and characterized. Nucleotide and deduced amino acid sequences of GmMIPS1 showed striking homology (80-99%) with other plant MIPS particularly with the dicots, Vigna radiata and Phaseolus vulgare. The protein sequence analysis of the predicted GmMIPS cDNA indicated the absence of signal peptide in the N-terminal region. To validate the expression of the GmMIPS1 coding gene, nucleotide sequence residues from 131 to 1,556 bp were amplified by high fidelity PCR and fused in frame to a 19 amino acid N-terminal region of 6X Histag in expression vector pET-28a (+). The E. coli strain BL21 (DE3) transformed with the recombinant plasmid resulted in the production of a 52 kDa fusion protein under optimized induction and expression conditions as confirmed by SDS-PAGE and Western blot analysis. Results of the present study suggested that down-regulation of GmMIPS1 using a seed specific promoter can be targeted as a great potential for development of low-phytate soybean without affecting the critical aspects of inositol metabolism in other tissues of the plant. Keywords: anti-nutrient, MIPS1, phytic acid, prokaryotic expression vector, soybean. Abbreviations: DAB-3,3´-diaminobenzidine tetrahydrochloride, IPTG- Isopropyl β-Dthiogalactopyranoside, MIPS- D-myo-inositol-3-phosphate synthase, ORF- open reading frame, PVDF- Polyvinylidene difluoride , SDS- PAGE- Sodium Dodecyl Sulphate- Polyacrylamide gel Electrophoresis, UTR- untranslated region. Introduction Soybean (Glycine max (L.) Merr.), one of the world's most important economic crops, has a steadily increasing agronomical value because of its high protein and vegetable oil content suitable for human and animal nutrition. While soybean is an important source of protein, its potential to provide energy and minerals has not fully reached in nonruminants animals including humans due to their inability to digest certain compounds such as phytates (Sebastian et al., 2000). Phytate (myo-inositol 1,2,3,4,5,6- hexakisphosphate), also known as phytic acid (PA) or phytin, is the major form of phosphorus (P) storage in seeds, comprising over 75–80% of the total P in plant seeds (Cosgrove 1966; Raboy et al., 2001). In soybean seeds, phytic acid accounts for up to 2% of the seed dry weight (Raboy et al. 1984). It begins to accumulate in seeds after the cellular phosphate levels have reached maximum levels and continues to increase linearly throughout seed development and seed filling (Raboy and Dickenson, 1987). It is usually deposited in protein bodies as a mixed salt (phytin), bound to mineral cations such as Fe3+, Ca2+, Mg2+, Zn2+ and K+ (Prattley and Stanley 1982, Lott 1984). Additionally, phytate in seed when broken down by the enzyme phytase, it becomes inorganic phosphate and myo-inositol, which are then available for seedling growth. Although an important storage molecule for growing seedlings, PA poses severe nutritional consequences as it acts as an antinutrient by forming indigestible complexes with minerals and proteins, decreasing the seeds nutritional quality. It chelates mineral cations, including calcium, zinc, magnesium and iron from the diet and affects the bioavailability of these essential minerals (Raboy et al., 2001). It also has the potential to bind charged amino acid residues of proteins resulting in a concomitant reduction of protein availability and digestibility. Also the excretion of unused P in the waste makes its way into the waterways causing environmental hazards. This antinutritional quality of phytate can be further extended to human health as it contributes to the iron deficiency suffered by over 2 billion people worldwide (Bouis 2000). The economic, nutritional, and environmental problems associated with phytate in animal or human feed can be reduced by developing low phytate soybean (Raboy 2007). The development of low phytic acid (lpa) crops is an important goal in genetic engineering programs aimed at improving the nutritional quality as well as at developing environment friendly and sustainable production. One approach for reduction of plant seed phytate levels involves the reduction of the expression of enzymes in the biosynthetic pathway of phytic acid. D-myo-inositol 3-phosphate synthase (MIPS, E.C. 5.5.1.4) catalyzes the NADH-dependent conversion of D-glucose 6-phosphate (G-6-P) to D-myo-inositol 3-phosphate (MIP) the first and the rate-limiting step of myo-inositol biosynthesis (Biswas et al., 1984; Loewus and Murthy, 2000). Further a stepwise phosphorylation (Fig. 4) of myo-inositol generates phytic acid. MIPS has been isolated and characterized from both prokaryotic and eukaryotic organisms. The structural gene coding for the MIPS was first identified in yeast (Donahue & Henry 1981; Majumder et al. 1981). Subsequently, MIPS coding sequences have been cloned and characterized from widely different organisms, including plants such as Spirodela polyrrhiza (Smart & Fleming 1993), Citrus paradisi (Abu-abied and Holland 1994), Arabidopsis thaliana (Johnson 1994; Johnson and Sussex 1995), Mesembryanthemum crystallinum (Ishitani et al. 1996), wild halophytic rice P. coarctata (Majee et al., 2004), Xerophyta viscosa (Majee et al. 2005), Passiflora edulis (Abreu & Aragao 2007), Cicer arietinum (Kaur et al., 2008), etc. Several plants have been found to possess multiple isoforms of MIPS enzyme, suggesting that each gene copy may be differentially controlled and expressed. Soybean contains four MIPS isoforms and one of the MIPS cDNAs (GmMIPS1) was shown to express mainly in developing seeds (Hegeman et al., 2001; Chappell et al., 2006). Using immunolocalization techniques, a specialized area of GmMIPS-1 expression has been identified in the outer integumentary layer during early soybean seed development (Chiera and Grabau, 2007). A number of genes homologous to GmMIPS1 have been reported till date and a “core catalytic structure” conserved across evolutionary divergent taxa has been identified (Majumdar et al., 2003). Down regulation of MIPS gene expression in seeds offer a potential approach for developing low-phytate soybean (Hitz and Sebastian, 1998). In the present study, we report the isolation, cloning and characterization of full length GmMIPS cDNAs from developing seeds of soybean and validation of its expression in Escherichia coli, especially with respect to its involvement in phytic acid biosynthesis. The fully functional GmMIPS1 gene can further be targeted for genetic manipulation by advanced gene silencing strategies to develop low phytate soybean seeds with improved nutritional value. Materials and methods Bacterial strains and plant materials Escherichia coli BL21 (DE3) and DH-5α strains were cultured on LB medium at 37oC. Cells containing recombinant plasmids, pGEMT-Easy and pET-28a(+) were supplemented with 100 mg ml-1 ampicillin and 50 mg ml-1 kanamycin. Soybean seeds (Glycine max) were collected from the Division of Genetics, Indian Agricultural Research Institute, New Delhi, India. Mercuric chloride (0.02%, 5 min.) sterilized seeds were sown in pots maintained under controlled environmental conditions at the National Phytotron Facility, I.A.R.I., New Delhi. The developing seeds (4 to 6 mm) were harvested and rapidly frozen in liquid nitrogen at -80oC. RNA isolation and RT-PCR amplification Total RNA was isolated from developing cotyledons (4 to 6mm seeds) of soybean samples (100 mg) using the RNeasy Plant Mini Kit (Qiagen) according to manufacturer’s instructions. Frozen plant tissues were homogenized using pestle and mortar with liquid nitrogen and one ml of Qiagen lysis buffer added per 100 mg of tissue in 2 ml micocentrifuge tubes. First strand of cDNA was synthesized from RNA by using oligo(dT) primer and reverse transcriptase from RevertAidTM H Minus first strand cDNA synthesis kit (Fermentas, Life Sciences). The full length cDNA for MIPS1 was amplified using oligonucleotide primers designed by BioEdit software based on the published soybean MIPS sequences (GenBank Accession Number AF293970) available in NCBI GenBank (forward primer: 5’-ATAGGATTCTCTTC TTTATTCCT-3´; reverse primer: 5´-TACACAAAATTATACTACATTCAT-3´). The PCR thermal cycling parameters used were 94○C denaturation for 4 min followed by 35 cyclesen_US
dc.identifier.urihttp://krishikosh.egranth.ac.in/handle/1/73018
dc.publisherIARI,Division of Biochemistryen_US
dc.subBiochemistry
dc.subjectanti-nutrient, MIPS1, phytic acid, prokaryotic expression vector, soybean. Abbreviations: DAB-3,3´-diaminobenzidine tetrahydrochloride, IPTG- Isopropyl β-Dthiogalactopyranoside, MIPS- D-myo-inositol-3-phosphate synthase, ORF- open reading frame, PVDF- Polyvinylidene difluoride , SDS- PAGE- Sodium Dodecyl Sulphate- Polyacrylamide gel Electrophoresis, UTR- untranslated region.en_US
dc.these.typePh.D
dc.titleCharacterization and expression of myo-inositol- 3- phosphate synthase (MIPS) gene in Glycine maxen_US
dc.title.alternativePh.D.en_US
dc.typeThesisen_US
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